6-amino-2,4-dihydropyrano [2,3-c] pyrazoles and methods of use

ABSTRACT

The present invention generally relates to 6-amino-2,4-dihydropyrano[2,3-c] pyrazoles as a ubiquitin specific protease 7 (USP7) inhibitor useful for the treatment of diseases mediated by malfunction of USP7, such as inflammation, cancer, and immunological disorders. The invention described herein also pertains to pharmaceutical compositions and methods for treating diseases mediated by malfunction of USP7, in mammals using compounds disclosed herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This present patent application is a divisional application of U.S.patent application Ser. No. 16/493,978, filed on Sep. 13, 2019, which isa national stage entry under 35 U.S.C. § 371(b) of InternationalApplication No. PCT/US18/24992, filed on Mar. 29, 2018, which relates toand claims the priority benefit of U.S. Provisional Application Ser. No.62/478,068, filed Mar. 29, 2017, the contents of which are herebyincorporated herein by reference in their entirety.

GOVERNMENT RIGHTS

This invention was made with government support under CA023168 awardedby the National Institutes of Health. The government has certain rightsin the invention.

TECHNICAL FIELD

The present disclosure generally relates to novel compounds as aubiquitin specific protease 7 (USP7) inhibitor useful for the treatmentof diseases mediated by USP7 malfunction, such as inflammation, cancerand immunological disorders, and in particular to6-amino-2,4-dihydropyrano[2,3-c] pyrazoles and methods of use. Theinvention described herein also pertains to pharmaceutical compositionsand methods for treating diseases mediated by malfunction of USP7, inmammals using compounds disclosed herein.

BACKGROUND

This section introduces aspects that may help facilitate a betterunderstanding of the disclosure. Accordingly, these statements are to beread in this light and are not to be understood as admissions about whatis or is not prior art.

Ubiquitin (Ub) is a highly conserved 76-amino acid protein that iscovalently attached through the formation of an isopeptide bond betweenthe ε-amino of a lysine residues of a protein substrate and theC-terminus of ubiquitin. The conjugation of ubiquitin to proteinsubstrates commonly signals for proteosomal degradation.

Ubiquitin-mediated degradation of regulatory proteins plays a crucialrole in numerous cellular processes, including cell-cycle progression,apoptosis, epigenetics and transcriptional regulation. Aberrations ofubiquitin-mediated processes have been linked to pathologicalconditions, including cancer, inflammation, and immunological diseases.While the attachment of ubiquitin to substrate proteins is catalyzed bythe sequential action of E1, E2 and E3 enzymes, the cleavage ofubiquitin is facilitated by specialized proteases called deubiquitinases(DUBs). The most studied of the DUBs is USP7, which has been shown toplay an important role in cancer through the regulation of the activityand cellular levels of tumor suppressor proteins such as p53, PTEN, andFOXO4.

The existence of a dynamic and complex interplay between p53 and itsregulatory proteins Mdm2 (corresponding to Hdm2 in humans, however wewill continue referring to Mdm2 for simplicity) and USP7 has beendemonstrated in several studies (Cummins, J. M. et al. Nature 2004, 428,6982). While the specifics of this interaction are still unclear, it isgenerally understood that in normal conditions cellular levels of p53are very low, due to its rapid proteosomal degradation, induced by theMdm2-catalyzed ubiquitination. USP7 participates in maintaining adynamic equilibrium through its ability to deubiquitinate Mdm2 and p53.

While both proteins compete for the same binding site on USP7, in theN-terminal TRAF-like domain, under normal, unstressed cellularconditions Mdm2 is the preferred substrate for USP7. Upon DNA damage andin stressed cells, ATP-dependent phosphorylation of Mdm2 lowers itsaffinity for USP7, resulting in a stress-induced degradation of Mdm2. Inthese condition USP7 preferentially deubiquitinates p53, resulting in anoverall stabilization of p53 (Zilfou, J. T. et al. Cold Spring HarborPerspectives in Biology 2009, 1 (5), a001883).

RNA interference studies have shown that USP7 silencing reduces cellsproliferation through induction of apoptosis. This phenotype wasobserved only in cancer cell lines with wild-type p53, including HCT116,MCF-7 and A549, and was associated with increased cellular levels of p53and increased degradation of Mdm2.

USP7 is also involved in processes of cellular proliferation that areindependent from p53. A representative example is the interaction withtranscription factors of the fork head box O (FOXO) family. Recentstudies have shown that following oxidative stress, FOXO4 undergoesubiquitination, and consequent translocation to the nucleus andactivation. The deubiquitynating activity of USP7 negatively affectsFOXO4 localization to the nucleus and transcriptional activity (van derHorst, A. et. al. Nat. Cell. Biol. 2006, 8 (10), 1064-1073).

Similarly, nuclear localization of the phosphatase and tensin homologue(PTEN) is crucial for its role as a tumor suppressor protein. Monoubiquitination induces translocation of PTEN to the nucleus and thereverse reaction, catalyzed by USP7, causes its nuclear exclusion,blocking its apoptotic potential in prostate cancer cells (Song, M. S.et. al. Nature 2008, 455 (7214), 813-817).

Overexpression of USP7 has been shown to correlate with tumoraggressiveness in several cancers (including prostate, non-small celllung cancer and glioma) and with short patient survival time and highmalignancy in non-small cell lung cancers. Taken together, the resultsof USP7 silencing experiments and the data showing the crucialinvolvement of USP7 in oncogenic pathways suggest that small moleculeinhibitors of USP7 have potential for anticancer therapies.

The FDA approval of the proteasome inhibitor Bortezomib has validatedthe approach of targeting protein degradation pathways and has alsodriven the development of small molecules targeting related enzymes. Oneof the limitations of Bortezomib therapy is the development ofresistance, which limits its long-term utility. Small moleculesinhibiting USP7 may overcome this resistance and indeed have showed toinduce apoptosis in multiple myeloma cells resistant to bortezomib.Inhibitors of USP7 have been discovered, but many of them have issuessuch as selectivity (due to the high homology of proteases, particularlyin the catalytic domain) or poor pharmacokinetic properties.

SUMMARY OF THE INVENTION

This present disclosure relates to compounds having a formula (I)

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,wherein

-   -   R¹ is hydrogen, an alkyl or an acyl;    -   R² is a heterocyclyl, cycloalkyl, cycloalkenyl,        cycloheteroalkyl, cycloheteroalkenyl, aryl, heteroaryl,        arylalkyl, arylalkenyl, or arylalkynyl, each of which is        optionally substituted;    -   R³, R⁴, R⁵, R⁶, and R⁷ represent five substituents each        independently selected from the group consisting of hydrogen,        halo, azido, cyano, nitro, hydroxy, amino, thio, and derivatives        thereof, and an acyl, alkyl, alkenyl, alkynyl, heteroalkyl,        heteroalkenyl, oxyalkyl, heteroalkynyl, a heterocyclyl,        cycloalkyl, cycloalkenyl, cycloheteroalkyl, cycloheteroalkenyl,        aryl, arylalkyl, arylalkenyl, and arylalkynyl, each of which is        optionally substituted; or any two adjacent substituents that        are taken together with the attached carbons to form an        optionally substituted heterocycle and each of other        substituents is defined as above; and    -   R⁸ is cyano or a carboxy ester.

In some preferred embodiments, this invention relates to compoundshaving a general formula (I) wherein R¹ is hydrogen.

In some embodiments, this invention relates to compounds having aformula general (I) wherein R² is an optionally substituted heterocycle.

In some preferred embodiments, this invention relates to compoundshaving a formula general (I), wherein R² is

In some preferred embodiments, this invention is related to compoundshaving a formula general (I) wherein R⁸ is cyano or a carboxy ester.

In some other embodiments, this invention is related to a pharmaceuticalcomposition comprising one or more compounds disclosed herein, or apharmaceutically acceptable salt thereof, together with one or morediluents, excipients or carriers.

In some other embodiments, this invention is related to a method fortreating diseases mediated by USP7 malfunction, such as inflammation,cancer and immunological disorders, comprising the step of administeringa therapeutically effective amount of one or more compounds disclosedherein, and one or more carriers, diluents, or excipients, to a patientin need of relief from said disorder.

In some other embodiments, this invention is related to a method fortreating diseases mediated by USP7 malfunction, such as inflammation,cancer and immunological disorders, comprising the step of administeringa therapeutically effective amount of one or more compounds disclosedherein in combination with one or more other compounds of the same ordifferent mode of action, and one or more carriers, diluents, orexcipients, to a patient in need of relief from said disorder.

These and other features, aspects and advantages of the presentinvention will become better understood with reference to the followingdescriptions and claims.

DETAILED DESCRIPTION

While the concepts of the present disclosure are illustrated anddescribed in detail in the figures and the description herein, resultsin the figures and their description are to be considered as exemplaryand not restrictive in character; it being understood that only theillustrative embodiments are shown and described and that all changesand modifications that come within the spirit of the disclosure aredesired to be protected.

The present disclosure generally relates to novel compounds as aubiquitin specific protease 7 (USP7) inhibitor useful for the treatmentof diseases mediated by USP7 malfunction, such as inflammation, cancerand immunological disorders, and in particular to6-amino-2,4-dihydropyrano[2,3-c] pyrazoles and methods of use. Theinvention described herein also pertains to pharmaceutical compositionsand methods for treating diseases mediated by malfunction of USP7, inmammals using compounds disclosed herein.

As used herein, the following terms and phrases shall have the meaningsset forth below. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art.

In the present disclosure the term “about” can allow for a degree ofvariability in a value or range, for example, within 10%, within 5%, orwithin 1% of a stated value or of a stated limit of a range. In thepresent disclosure the term “substantially” can allow for a degree ofvariability in a value or range, for example, within 90%, within 95%,99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more of a statedvalue or of a stated limit of a range.

In this document, the terms “a,” “an,” or “the” are used to include oneor more than one unless the context clearly dictates otherwise. The term“or” is used to refer to a nonexclusive “or” unless otherwise indicated.In addition, it is to be understood that the phraseology or terminologyemployed herein, and not otherwise defined, is for the purpose ofdescription only and not of limitation. Any use of section headings isintended to aid reading of the document and is not to be interpreted aslimiting. Further, information that is relevant to a section heading mayoccur within or outside of that particular section. Furthermore, allpublications, patents, and patent documents referred to in this documentare incorporated by reference herein in their entirety, as thoughindividually incorporated by reference. In the event of inconsistentusages between this document and those documents so incorporated byreference, the usage in the incorporated reference should be consideredsupplementary to that of this document; for irreconcilableinconsistencies, the usage in this document controls.

The term “substituted” as used herein refers to a functional group inwhich one or more hydrogen atoms contained therein are replaced by oneor more non-hydrogen atoms. The term “functional group” or “substituent”as used herein refers to a group that can be or is substituted onto amolecule. Examples of substituents or functional groups include, but arenot limited to, a halogen (e.g., F, Cl, Br, and I); an oxygen atom ingroups such as hydroxyl groups, alkoxy groups, aryloxy groups,aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups includingcarboxylic acids, carboxylates, and carboxylate esters; a sulfur atom ingroups such as thiol groups, alkyl and aryl sulfide groups, sulfoxidegroups, sulfone groups, sulfonyl groups, and sulfonamide groups; anitrogen atom in groups such as amines, azides, hydroxylamines, cyano,nitro groups, N-oxides, hydrazides, and enamines; and other heteroatomsin various other groups.

The term “alkyl” as used herein refers to substituted or unsubstitutedstraight chain and branched alkyl groups and cycloalkyl groups havingfrom 1 to about 20 carbon atoms (C₁-C₂₀), 1 to 12 carbons (C₁-C₁₂), 1 to8 carbon atoms (C₁-C₈), or, in some embodiments, from 1 to 6 carbonatoms (C₁-C₆). Examples of straight chain alkyl groups include thosewith from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl,n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branchedalkyl groups include, but are not limited to, isopropyl, iso-butyl,sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups.As used herein, the term “alkyl” encompasses n-alkyl, isoalkyl, andanteisoalkyl groups as well as other branched chain forms of alkyl.Representative substituted alkyl groups can be substituted one or moretimes with any of the groups listed herein, for example, amino, hydroxy,cyano, carboxy, nitro, thio, alkoxy, and halogen groups.

The term “alkenyl” as used herein refers to substituted or unsubstitutedstraight chain and branched divalent alkenyl and cycloalkenyl groupshaving from 2 to 20 carbon atoms (C₂-C₂₀), 2 to 12 carbons (C₂-C₁₂), 2to 8 carbon atoms (C₂-C₈) or, in some embodiments, from 2 to 4 carbonatoms (C₂-C₄) and at least one carbon-carbon double bond. Examples ofstraight chain alkenyl groups include those with from 2 to 8 carbonatoms such as —CH═CH—, —CH═CHCH₂—, and the like. Examples of branchedalkenyl groups include, but are not limited to, —CH═C(CH₃)— and thelike.

An alkynyl group is the fragment, containing an open point of attachmenton a carbon atom that would form if a hydrogen atom bonded to a triplybonded carbon is removed from the molecule of an alkyne. The term“hydroxyalkyl” as used herein refers to alkyl groups as defined hereinsubstituted with at least one hydroxyl (—OH) group.

The term “cycloalkyl” as used herein refers to substituted orunsubstituted cyclic alkyl groups such as, but not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, andcyclooctyl groups. In some embodiments, the cycloalkyl group can have 3to about 8-12 ring members, whereas in other embodiments the number ofring carbon atoms ranges from 3 to 4, 5, 6, or 7. In some embodiments,cycloalkyl groups can have 3 to 6 carbon atoms (C₃-C₆). Cycloalkylgroups further include polycyclic cycloalkyl groups such as, but notlimited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, andcarenyl groups, and fused rings such as, but not limited to, decalinyl,and the like.

The term “acyl” as used herein refers to a group containing a carbonylmoiety wherein the group is bonded via the carbonyl carbon atom. Thecarbonyl carbon atom is also bonded to another carbon atom, which can bepart of a substituted or unsubstituted alkyl, aryl, aralkyl cycloalkyl,cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl,heteroarylalkyl group or the like. In the special case wherein thecarbonyl carbon atom is bonded to a hydrogen, the group is a “formyl”group, an acyl group as the term is defined herein. An acyl group caninclude 0 to about 12-40, 6-10, 1-5 or 2-5 additional carbon atomsbonded to the carbonyl group. An acryloyl group is an example of an acylgroup. An acyl group can also include heteroatoms within the meaninghere. A nicotinoyl group (pyridyl-3-carbonyl) is an example of an acylgroup within the meaning herein. Other examples include acetyl, benzoyl,phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl groups and thelike. When the group containing the carbon atom that is bonded to thecarbonyl carbon atom contains a halogen, the group is termed a“haloacyl” group. An example is a trifluoroacetyl group.

The term “aryl” as used herein refers to substituted or unsubstitutedcyclic aromatic hydrocarbons that do not contain heteroatoms in thering. Thus aryl groups include, but are not limited to, phenyl,azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl,triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl,anthracenyl, and naphthyl groups. In some embodiments, aryl groupscontain about 6 to about 14 carbons (C₆-C₁₄) or from 6 to 10 carbonatoms (C₆-C₁₀) in the ring portions of the groups. Aryl groups can beunsubstituted or substituted, as defined herein. Representativesubstituted aryl groups can be mono-substituted or substituted more thanonce, such as, but not limited to, 2-, 3-, 4-, 5-, or 6-substitutedphenyl or 2-8 substituted naphthyl groups, which can be substituted withcarbon or non-carbon groups such as those listed herein.

The term “aralkyl” and “arylalkyl” as used herein refers to alkyl groupsas defined herein in which a hydrogen or carbon bond of an alkyl groupis replaced with a bond to an aryl group as defined herein.Representative aralkyl groups include benzyl and phenylethyl groups andfused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Aralkenylgroups are alkenyl groups as defined herein in which a hydrogen orcarbon bond of an alkyl group is replaced with a bond to an aryl groupas defined herein.

The term “heterocyclyl” as used herein refers to substituted orunsubstituted aromatic and non-aromatic ring compounds containing 3 ormore ring members, of which, one or more is a heteroatom such as, butnot limited to, B, N, O, and S. Thus, a heterocyclyl can be acycloheteroalkyl, or a heteroaryl, or if polycyclic, any combinationthereof. In some embodiments, heterocyclyl groups include 3 to about 20ring members, whereas other such groups have 3 to about 15 ring members.In some embodiments, heterocyclyl groups include heterocyclyl groupsthat include 3 to 8 carbon atoms (C₃-C₈), 3 to 6 carbon atoms (C₃-C₆) or6 to 8 carbon atoms (C₆-C₈).

A heteroaryl ring is an embodiment of a heterocyclyl group. The phrase“heterocyclyl group” includes fused ring species, such as those fusedaromatic and non-aromatic groups. Representative heterocyclyl groupsinclude, but are not limited to pyrrolidinyl, azetidinyl, piperidynyl,piperazinyl, morpholinyl, chromanyl, indolinonyl, isoindolinonyl,furanyl, pyrrolidinyl, pyridinyl, pyrazinyl, pyrimidinyl, triazinyl,thiophenyl, tetrahydrofuranyl, pyrrolyl, oxazolyl, oxadiazolyl,imidazolyl, triazyolyl, tetrazolyl, benzoxazolinyl, benzothiazolinyl,and benzimidazolinyl groups.

The term “heterocyclylalkyl” as used herein refers to alkyl groups asdefined herein in which a hydrogen or carbon bond of an alkyl group asdefined herein is replaced with a bond to a heterocyclyl group asdefined herein. Representative heterocyclylalkyl groups include, but arenot limited to, furan-2-yl methyl, furan-3-yl methyl, pyridine-3-ylmethyl, tetrahydrofuran-2-yl methyl, and indol-2-yl propyl.

The term “heteroarylalkyl” as used herein refers to alkyl groups asdefined herein in which a hydrogen or carbon bond of an alkyl group isreplaced with a bond to a heteroaryl group as defined herein.

The term “alkoxy” as used herein refers to an oxygen atom connected toan alkyl group, including a cycloalkyl group, as are defined herein.Examples of linear alkoxy groups include but are not limited to methoxy,ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples ofbranched alkoxy include but are not limited to isopropoxy, sec-butoxy,tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclicalkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy,cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can furtherinclude double or triple bonds, and can also include heteroatoms. Forexample, an allyloxy group is an alkoxy group within the meaning herein.A methoxyethoxy group is also an alkoxy group within the meaning herein,as is a methylenedioxy group in a context where two adjacent atoms of astructure are substituted therewith.

The term “amine” as used herein refers to primary, secondary, andtertiary amines having, e.g., the formula N(group)₃ wherein each groupcan independently be H or non-H, such as alkyl, aryl, and the like.Amines include but are not limited to R—NH₂, for example, alkylamines,arylamines, alkylarylamines; R₂NH wherein each R is independentlyselected, such as dialkylamines, diarylamines, aralkylamines,heterocyclylamines and the like; and R₃N wherein each R is independentlyselected, such as trialkylamines, dialkylarylamines, alkyldiarylamines,triarylamines, and the like. The term “amine” also includes ammoniumions as used herein.

The term “amino group” as used herein refers to a substituent of theform —NH₂, —NHR, —NR₂, —NR₃ ⁺, wherein each R is independently selected,and protonated forms of each, except for —NR₃ ⁺, which cannot beprotonated. Accordingly, any compound substituted with an amino groupcan be viewed as an amine. An “amino group” within the meaning hereincan be a primary, secondary, tertiary, or quaternary amino group. An“alkylamino” group includes a monoalkylamino, dialkylamino, andtrialkylamino group.

The terms “halo,” “halogen,” or “halide” group, as used herein, bythemselves or as part of another substituent, mean, unless otherwisestated, a fluorine, chlorine, bromine, or iodine atom.

The term “haloalkyl” group, as used herein, includes mono-halo alkylgroups, poly-halo alkyl groups wherein all halo atoms can be the same ordifferent, and per-halo alkyl groups, wherein all hydrogen atoms arereplaced by halogen atoms, such as fluoro. Examples of halo alkylinclude trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl,1,3-dibromo-3,3-difluoropropyl, perfluorobutyl, —CF(CH₃)₂ and the like.

The term “optionally substituted,” or “optional substituents,” as usedherein, means that the groups in question are either unsubstituted orsubstituted with one or more of the substituents specified. When thegroups in question are substituted with more than one substituent, thesubstituents may be the same or different. When using the terms“independently,” “independently are,” and “independently selected from”mean that the groups in question may be the same or different. Certainof the herein defined terms may occur more than once in the structure,and upon such occurrence each term shall be defined independently of theother.

The compounds described herein may contain one or more chiral centers,or may otherwise be capable of existing as multiple stereoisomers. It isto be understood that in one embodiment, the invention described hereinis not limited to any particular stereochemical requirement, and thatthe compounds, and compositions, methods, uses, and medicaments thatinclude them may be optically pure, or may be any of a variety ofstereoisomeric mixtures, including racemic and other mixtures ofenantiomers, other mixtures of diastereomers, and the like. It is alsoto be understood that such mixtures of stereoisomers may include asingle stereochemical configuration at one or more chiral centers, whileincluding mixtures of stereochemical configuration at one or more otherchiral centers.

Similarly, the compounds described herein may include geometric centers,such as cis, trans, E, and Z double bonds. It is to be understood thatin another embodiment, the invention described herein is not limited toany particular geometric isomer requirement, and that the compounds, andcompositions, methods, uses, and medicaments that include them may bepure, or may be any of a variety of geometric isomer mixtures. It isalso to be understood that such mixtures of geometric isomers mayinclude a single configuration at one or more double bonds, whileincluding mixtures of geometry at one or more other double bonds.

As used herein, the term “salts” and “pharmaceutically acceptable salts”refer to derivatives of the disclosed compounds wherein the parentcompound is modified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic groups such as amines; and alkalior organic salts of acidic groups such as carboxylic acids.Pharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. For example,such conventional non-toxic salts include those derived from inorganicacids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,and nitric; and the salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, andisethionic, and the like.

Pharmaceutically acceptable salts can be synthesized from the parentcompound which contains a basic or acidic moiety by conventionalchemical methods. In some instances, such salts can be prepared byreacting the free acid or base forms of these compounds with astoichiometric amount of the appropriate base or acid in water or in anorganic solvent, or in a mixture of the two; generally, nonaqueous medialike ether, ethyl acetate, ethanol, isopropanol, or acetonitrile arepreferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, the disclosure of which is hereby incorporated by reference.

The term “solvate” means a compound, or a salt thereof, that furtherincludes a stoichiometric or non-stoichiometric amount of solvent boundby non-covalent intermolecular forces. Where the solvent is water, thesolvate is a hydrate.

The term “prodrug” means a derivative of a compound that can hydrolyze,oxidize, or otherwise react under biological conditions (in vitro or invivo) to provide an active compound, particularly a compound of theinvention. Examples of prodrugs include, but are not limited to,derivatives and metabolites of a compound of the invention that includebiohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzableesters, biohydrolyzable carbamates, biohydrolyzable carbonates,biohydrolyzable ureides, and biohydrolyzable phosphate analogues.Specific prodrugs of compounds with carboxyl functional groups are thelower alkyl esters of the carboxylic acid. The carboxylate esters areconveniently formed by esterifying any of the carboxylic acid moietiespresent on the molecule. Prodrugs can typically be prepared usingwell-known methods, such as those described by Burger's MedicinalChemistry and Drug Discovery 6th ed. (Donald J. Abraham ed., 2001,Wiley) and Design and Application of Prodrugs (H. Bundgaard ed., 1985,Harwood Academic Publishers GmbH).

Further, in each of the foregoing and following embodiments, it is to beunderstood that the formulae include and represent not only allpharmaceutically acceptable salts of the compounds, but also include anyand all hydrates and/or solvates of the compound formulae or saltsthereof. It is to be appreciated that certain functional groups, such asthe hydroxy, amino, and like groups form complexes and/or coordinationcompounds water and/or various solvents, in the various physical formsof the compounds. Accordingly, the above formulae are to be understoodto include and represent those various hydrates and/or solvates. In eachof the foregoing and following embodiments, it is also to be understoodthat the formulae include and represent each possible isomer, such asstereoisomers, tautomers and geometric isomers, both individually and inany and all possible mixtures. In each of the foregoing and followingembodiments, it is also to be understood that the formulae include andrepresent any and all crystalline forms, partially crystalline forms,and non-crystalline and/or amorphous forms of the compounds.

The term “pharmaceutically acceptable carrier” is art-recognized andrefers to a pharmaceutically-acceptable material, composition orvehicle, such as a liquid or solid filler, diluent, excipient, solventor encapsulating material, involved in carrying or transporting anysubject composition or component thereof. Each carrier must be“acceptable” in the sense of being compatible with the subjectcomposition and its components and not injurious to the patient. Someexamples of materials which may serve as pharmaceutically acceptablecarriers include: (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)talc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations.

As used herein, the term “administering” includes all means ofintroducing the compounds and compositions described herein to thepatient, including, but are not limited to, oral (po), intravenous (iv),intramuscular (im), subcutaneous (sc), transdermal, inhalation, buccal,ocular, sublingual, vaginal, rectal, and the like. The compounds andcompositions described herein may be administered in unit dosage formsand/or formulations containing conventional nontoxic pharmaceuticallyacceptable carriers, adjuvants, and vehicles.

Illustrative formats for oral administration include tablets, capsules,elixirs, syrups, and the like. Illustrative routes for parenteraladministration include intravenous, intraarterial, intraperitoneal,epidural, intraurethral, intrasternal, intramuscular and subcutaneous,as well as any other art recognized route of parenteral administration.

Illustrative means of parenteral administration include needle(including microneedle) injectors, needle-free injectors and infusiontechniques, as well as any other means of parenteral administrationrecognized in the art. Parenteral formulations are typically aqueoussolutions which may contain excipients such as salts, carbohydrates andbuffering agents (preferably at a pH in the range from about 3 to about9), but, for some applications, they may be more suitably formulated asa sterile non-aqueous solution or as a dried form to be used inconjunction with a suitable vehicle such as sterile, pyrogen-free water.The preparation of parenteral formulations under sterile conditions, forexample, by lyophilization, may readily be accomplished using standardpharmaceutical techniques well known to those skilled in the art.Parenteral administration of a compound is illustratively performed inthe form of saline solutions or with the compound incorporated intoliposomes. In cases where the compound in itself is not sufficientlysoluble to be dissolved, a solubilizer such as ethanol can be applied.

The dosage of each compound of the claimed combinations depends onseveral factors, including: the administration method, the condition tobe treated, the severity of the condition, whether the condition is tobe treated or prevented, and the age, weight, and health of the personto be treated. Additionally, pharmacogenomic (the effect of genotype onthe pharmacokinetic, pharmacodynamic or efficacy profile of atherapeutic) information about a particular patient may affect thedosage used.

It is to be understood that in the methods described herein, theindividual components of a co-administration, or combination can beadministered by any suitable means, contemporaneously, simultaneously,sequentially, separately or in a single pharmaceutical formulation.Where the co-administered compounds or compositions are administered inseparate dosage forms, the number of dosages administered per day foreach compound may be the same or different. The compounds orcompositions may be administered via the same or different routes ofadministration. The compounds or compositions may be administeredaccording to simultaneous or alternating regimens, at the same ordifferent times during the course of the therapy, concurrently individed or single forms.

The term “therapeutically effective amount” as used herein, refers tothat amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician, which includes alleviation of the symptoms of thedisease or disorder being treated. In one aspect, the therapeuticallyeffective amount is that which may treat or alleviate the disease orsymptoms of the disease at a reasonable benefit/risk ratio applicable toany medical treatment. However, it is to be understood that the totaldaily usage of the compounds and compositions described herein may bedecided by the attending physician within the scope of sound medicaljudgment. The specific therapeutically-effective dose level for anyparticular patient will depend upon a variety of factors, including thedisorder being treated and the severity of the disorder; activity of thespecific compound employed; the specific composition employed; the age,body weight, general health, gender and diet of the patient: the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidentally with the specific compound employed; andlike factors well known to the researcher, veterinarian, medical doctoror other clinician of ordinary skill.

Depending upon the route of administration, a wide range of permissibledosages are contemplated herein, including doses falling in the rangefrom about 1 μg/kg to about 1 g/kg. The dosages may be single ordivided, and may administered according to a wide variety of protocols,including q.d. (once a day), (twice a day), t.i.d. (three times a day),or even every other day, once a week, once a month, once a quarter, andthe like. In each of these cases it is understood that thetherapeutically effective amounts described herein correspond to theinstance of administration, or alternatively to the total daily, weekly,month, or quarterly dose, as determined by the dosing protocol.

In addition to the illustrative dosages and dosing protocols describedherein, it is to be understood that an effective amount of any one or amixture of the compounds described herein can be determined by theattending diagnostician or physician by the use of known techniquesand/or by observing results obtained under analogous circumstances. Indetermining the effective amount or dose, a number of factors areconsidered by the attending diagnostician or physician, including, butnot limited to the species of mammal, including human, its size, age,and general health, the specific disease or disorder involved, thedegree of or involvement or the severity of the disease or disorder, theresponse of the individual patient, the particular compoundadministered, the mode of administration, the bioavailabilitycharacteristics of the preparation administered, the dose regimenselected, the use of concomitant medication, and other relevantcircumstances.

The term “patient” includes human and non-human animals such ascompanion animals (dogs and cats and the like) and livestock animals.Livestock animals are animals raised for food production. The patient tobe treated is preferably a mammal, in particular a human being.

In some illustrative embodiments, the invention relates to a compound offormula (I)

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,wherein

-   -   R¹ is hydrogen, an alkyl or an acyl;    -   R² is a heterocyclyl, cycloalkyl, cycloalkenyl,        cycloheteroalkyl, cycloheteroalkenyl, aryl, heteroaryl,        arylalkyl, arylalkenyl, or arylalkynyl, each of which is        optionally substituted;    -   R³, R⁴, R⁵, R⁶, and R⁷ represent five substituents each        independently selected from the group consisting of hydrogen,        halo, azido, cyano, nitro, hydroxy, amino, thio, and derivatives        thereof, and an acyl, alkyl, alkenyl, alkynyl, heteroalkyl,        heteroalkenyl, oxyalkyl, heteroalkynyl, a heterocyclyl,        cycloalkyl, cycloalkenyl, cycloheteroalkyl, cycloheteroalkenyl,        aryl, arylalkyl, arylalkenyl, and arylalkynyl, each of which is        optionally substituted; or any two adjacent substituents that        are taken together with the attached carbons to form an        optionally substituted heterocycle and each of other        substituents is defined as above; and    -   R⁸ is cyano or a carboxy ester.

In some illustrative embodiments, the invention relates to a compound offormula (I), wherein R¹ is hydrogen, an acyl or an alkyl.

In some illustrative embodiments, the invention relates to a compound offormula (I), wherein R² is an optionally substituted heterocycle.

In some preferred embodiments, the invention relates to a compound offormula (I), wherein R² is

In some illustrative embodiments, the invention relates to a compound offormula (I), wherein R¹ is hydrogen, an acyl or an alkyl; and R² is

In some illustrative embodiments, the invention relates to a compound offormula (I), wherein R⁴ or R⁶ is an alkyl, halo, or haloalkyl.

In some illustrative embodiments, the invention relates to a compound offormula (I), wherein R⁵ is hydroxyl, amino, nitro, halo, cyano, azido,an alkyl, alkenyl, alkynyl, alkyloxy, alkenyloxy, alkynyl, alkylamino,alkenylamino, oxyalkyl, or alkynylamino.

In some preferred embodiments, the invention relates to a compound offormula (I), wherein R⁵ is oxyalkyl.

In some illustrative embodiments, the invention relates to a compound offormula (I), wherein R⁵ is oxyalkyl; and R⁶ is halo, alkyl, cyano, orhaloalky.

In some illustrative embodiments, the invention relates to a compound offormula (I), wherein R⁴ is hydrogen; R⁵ is oxyalkyl; and R⁶ is halo,alkyl, cyano, or haloalky.

In some illustrative embodiments, the invention relates to a compound offormula (I), wherein R² is an optionally substituted heterocycle; R⁴ ishydrogen; R⁵ is oxyalkyl; and R⁶ is halo, alkyl, cyano, or haloalkyl.

In some illustrative embodiments, the invention relates to a compound offormula (I), wherein R¹ and R⁴ are hydrogen; R² is an optionallysubstituted heterocycle; R⁵ is oxyalkyl; and R⁶ is halo, alkyl, cyano,or haloalky.

In some illustrative embodiments, the invention relates to a compound offormula (I), wherein R⁴ or R⁶ is an alkyl, halo, or haloalkyl, and R⁵ isan oxyalkyl or aminoalkyl.

In some illustrative embodiments, the invention relates to a compound offormula (I), wherein R⁸ is a carboxy ester.

In some illustrative embodiments, the invention relates to a compound offormula (I), wherein said compound is

In some illustrative embodiments, the invention relates to a compound offormula (I), wherein said compound is

In some illustrative embodiments, the invention relates to a compound offormula (I), wherein said compound is

In some other illustrative embodiments, the invention relates to apharmaceutical composition comprising one or more compounds disclosedherein, or a pharmaceutically acceptable salt thereof, together with oneor more diluents, excipients or carriers.

In some other embodiments, this invention is related to a method fortreating diseases mediated by USP7 malfunction, such as inflammation,cancer and immunological disorders, wherein the method comprises thestep of administering a therapeutically effective amount of one or morecompounds disclosed herein, and one or more carriers, diluents, orexcipients, to a patient in need of relief from said disorder.

In some other embodiments, this invention is related to a method fortreating diseases mediated by USP7 malfunction, such as inflammation,cancer and immunological disorders, wherein the method comprises thestep of administering a therapeutically effective amount of one or morecompounds disclosed herein in combination with one or more othercompounds of the same or different mode of action, and one or morecarriers, diluents, or excipients, to a patient in need of relief fromsaid disorder.

In addition, it is appreciated herein that the compounds describedherein may be used in combination with other compounds that areadministered to treat other symptoms of cancer, such as compoundsadministered to relieve pain, nausea, vomiting, and the like.

The following non-limiting exemplary embodiments are included herein tofurther illustrate the invention. These exemplary embodiments are notintended and should not be interpreted to limit the scope of theinvention in any way. It is also to be understood that numerousvariations of these exemplary embodiments are contemplated herein.

The synthesis began with the reaction of differently substituted methylketones 1a-g with diethyl carbonate and NaH in THF to yield arylsubstituted β-ketoesters 2a-g (Scheme 1). These were then reacted withhydrazine or methyl hydrazine in refluxing ethanol to generatepyrazolones 4a-h. The regiochemistry of the resulting pyrazolones andthe mechanism of the reaction were studied in detail by Katrinsky(Katrizky, A. R., et al., J. Chem. Soc, Perkin Trans. 2, 1987, (8), 969)and confirmed by our 2D NMR analysis of the final dihydropyrano [2,3-c]pyrazoles 6a and 9a. The pyrazolones were then used in a three-componentreaction with malononitrile or ethylcyanoacetate and differentlysubstituted aldehydes to generate compounds 6a-v, 7a-o and 8 as racemicmixtures (Scheme 2). The three-component reaction was found a convenientmethod for the preparation of a diverse library of compounds in highpurities, and was preferred to the four-component protocol.

The scaffold was further functionalized to investigate the effect ofeach substituent on the activity (Scheme 3). Alkylation of the aminogroup at C-6 was obtained in a two steps-protocol which includes firstthe treatment of compound 6a or 9a with triethyl orthoformate in glacialacetic acid at 110° C. and then the resulting imidate 10 or 12 wasreduced to the methyl ethyl ether derivative 11 or to the methylanalogue 13, using respectively 1.5 or 3 equiv. of sodium borohydrate.

wherein:

Compound # R¹ R² 4a 2-thiophenyl H 4b 3-thiophenyl H 4c 2-pyridinyl H 4d3-pyridinyl H 4e 4-chlorophenyl H 4f 3(N-methyl) pyrrolyl H 4g3-(2,5-dimethylthiophenyl) H 4h 2-thiophenyl Me

Acetylation of 2-N was done by treating compound 6a with aceticanhydride at 110° C. to yield the acetyl derivative 14. Removal of NH₂occurred in a one-pot reductive deamination protocol, which includesgeneration of the diazonium salt by reacting compound 6a with tert-butylnitrite in DMF from −30° C. to room temperature, followed by thereduction of the intermediate with sodium borohydride to yield compound15 in 31% yield.

Other attempts to modify the scaffold, starting from 6a or 9a were notsuccessful. For example, the hydrolysis of the nitrile (in acidic orbasic conditions), or its conversion to the tetrazole (using sodiumazide, ammonium chloride and a polar solvent such as DMF or ethanol, attemperatures of 80° C. or 120° C.), resulted in loss of the scaffold orin a retrocondensation, which yielded pyrazolone 4a and arylidenemalononitrile.

Biological Assay and Results

The lyophilized compounds were re-suspended in 100% DMSO to a stockconcentration of 10 mM and stored at −20° C. Where Rate_(sample) is theinitial slope of the progress curve as measured in ArbitraryFluorescence Units per second of USP7 in the presence of compound.Rate_(pos) is the initial slope of USP7 without a compound present andRate_(neg) is the baseline of substrate hydrolysis without USP7 present.

The percent inhibition of USP7 at 100 μM of each compound was determinedprior to the determination of IC₅₀ values. The final concentration ofsubstrate was held constant at 200 nM and USP7 was held constant at afinal concentration of 1 nM in Assay Buffer (50 mM Tris pH 7.5, 5 mMDTT, 0.1 mg/mL BSA, and 0.01% Triton X-100). From the 10 mM stock, eachcompound to be tested was diluted to a working concentration of 3 mM in100% DMSO. The assay was performed as follows: 1 μL of the working stockof compound was added to a Costar 96 half-volume black plate to which 15μL of USP7 was added. Plates were gently mixed and incubated at roomtemperature for five minutes. To initiate the reaction, 15 μL ofUb-Rho110 was added. Each assay was measured in triplicate. A negativecontrol of Ub-Rho110 alone was measured to evaluate the background rate.Control reactions of USP7 without compound (DMSO only) were included tomeasure the rate of the uninhibited USP7 reaction. All reactionscontained a final concentration of 3% DMSO. The reaction progress wasmeasured as a filter based assay in 10-second intervals for a total of30 minutes at an excitation wavelength of 485 nm and an emissionwavelength of 528 nm. The percent inhibition was calculated usingEquation (1).

$\begin{matrix}{{\% {Inhibition}} = \lbrack {1 - \frac{{Rate}_{sample} - {Rate}_{neg}}{{Rate}_{pos} - {Rate}_{neg}}} \rbrack} & ( {{Equation}\mspace{14mu} 1} ) \\{{\% {Inhibition}} = \frac{{Maximum}\% {Inhibition}*\lbrack{Inhibitor}\rbrack}{\lbrack{Inhibitor}\rbrack + {IC}_{50}}} & ( {{Equation}\mspace{14mu} 2} )\end{matrix}$

The biochemical activity of the compounds synthesized is reported inTable 1 as a measure of their maximum % inhibition at concentration of100 μM. IC₅₀ values were determined for the compounds that showed apercent inhibition ≥40% at 100 μM. The IC₅₀ values were determined with11 compound concentrations ranging from 6 mM to 5.8 μM in two-foldserial dilutions in 100% DMSO. The assay design was identical to asdescribed above. The percent inhibition was calculated using Equation 1and fit to Equation 2 by use of the Enzyme Kinetics Module of Sigma Plot(v13:Systat Software Inc.), to determine the IC₅₀ value.

TABLE 1 Compound % inhibition Compound % inhibition Entry No. @ 100 μMIC₅₀ (μM) Entry No. @ 100 μM IC₅₀ (μM) 1 6a 72 ± 3 3.7 ± 0.4 2 6b 10 ± 7NA 3 6c 0 NA 4 6d 0 NA 5 6e 0 NA 6 6f 31 ± 6 NA 7 6g 57 ± 2 33 ± 5 8 6h38 ± 6 NA 9 6i 63 ± 1 NA 10 61 26 ± 3 NA 11 6m 32 ± 5 NA 12 6n 84 ± 24.5 ± 0.6 13 6o 45 ± 9 13 ± 3 14 6p 62 ± 20 NA 15 6q 92 ± 2 11.0 ± 0.716 6r 95 ± 4 1.4± 0.2 17 6s 32 ± 13 7 ± 2 18 6t 55 ± 3 7 ± 2 19 6u 85 ±1 1.8 ± 0.1 20 6v 93 ± 4 0.75 ± 0.06 21 7a 72 ± 4 8.1± 1 22 7b 71 ± 35.6 ± 0.7 23 7c 89 ± 2 7.9 ± 0.4 24 7d 89 ± 2 4.4 tested at 150 μM 25 7e82 ± 9 24 ± 5 26 7f 83 ± 3 7.7 ± 0.9 27 7g 34 ± 6 NA 28 7h 66 ± 3 7.5 ±0.9 29 7i 69 ± 3 15 ± 2 30 71 88.0 ± 0.09 1.91 ± 0.8 31 7m 89 ± 1 1.31 ±0.06 32 7n 32 ± 3 NA 33 70 29 ± 6 NA 34 9a 0 NA 35 9b 0 NA 36 11 0 NA 3713 47 ± 1 NA 38 14 85 ± 2 0.53 ± 0.05 39 15 0 NA

Compound Examples

Analytical thin layer chromatography (tic or TLC) was carried out onsilica gel plates (silica gel 60 F254). Eluted plates were visualized byexposure to ultraviolet light and then by staining with an ethanolicsolution of phosphomolybdic acid. The products were isolated andpurified using a flash chromatography system, with a mixture of hexanesand ethyl acetate as the eluent. The proton (¹H), ¹³C, HMBC and HSQC NMRspectra were taken on an 800 or a 500 MHz NMR spectrophotometer.Chemical shifts (6) are expressed in ppm relative to chloroform ortetramethylsilane or dmso-proton NMR coupling constants (J) areexpressed in Hz, and multiplicity is described as follows: s=singlet;d=doublet; t=triplet; q=quartet; ABq=AB quartet; quint=quintet;sext=sextet; sept=septet; br=broad; m=multiplet; dd=doublet of doublets;dt=doublet of triplets; dsept=doublet of septets; td=triplet ofdoublets; ddd=doublet of doublet of doublets. High-resolution massspectra (HRMS) and electrospray (ESI) experiments were performed with atime-of-flight (TOF) mass detector. HPLC analysis was performed onAgilent 1100. Specific conditions used are indicated for each compound.

General Method for the Preparation of β-Ketoesters

A three necks-round bottom flask, equipped with addition funnel,nitrogen inlet and temperature probe was charged with anhydrous THF andNaH (60% dispersion in mineral oil, 2 equiv. c=1.25 M, concentrationreferred to moles of NaH). The suspension was stirred at roomtemperature for 10 min and then a THF solution of 2-acetylthiophene (25mmol, 1 equiv. c=0.62 M) was added dropwise over a period of 20 min. Aslight (4-5° C.) increase of the temperature was observed during theaddition, and then the reaction mixture was warmed to 35° C. and stirredfor 30 min. A THF solution of diethyl carbonate (50 mmol, 2 equiv.,c=1.70 M) was added over a period of 1 hour. After one additional hour,the reaction mixture was cooled down to −10° C. and quenched with slowaddition of water (5-10 ml), and then glacial acetic (3 ml) was added.The mixture was stirred for 20 min and then warmed to room temperature.The organic layer was separated and the aqueous layer was extracted withethyl acetate (3×50 ml). The combined organic layers were washed withbrine, dried with anhydrous Na₂SO₄ and concentrated under reducedpressure. Purification was done by automated flash chromatography usingsilica gel column and a mixture of hexanes and ethyl acetate as eluent.Hexanes (100%) was used to elute the excess of diethyl carbonate, andthe amount of ethyl acetate was progressively increased from 20% to 50%to elute the title compound.

Ethyl 3-oxo-3-(thiophen-2-yl)propanoate (2a) (81% yield)

Proton NMR (800 MHz, Chloroform-d) δ 7.75 (dd, J=3.7, 1.3 Hz, 1H), 7.70(dd, J=4.9, 1.3 Hz, 1H), 7.15 (dd, J=4.9, 3.7 Hz, 1H), 4.22 (q, J=7.1Hz, 2H), 3.92 (s, 2H), 1.27 (t, J=7.1 Hz, 3H). ¹³C NMR (200 MHz, CDCl₃)δ 185.1, 167.1, 143.4, 135.1, 133.4, 128.5, 61.8, 46.7, 14.2.

Ethyl 3-oxo-3-(thiophen-3-yl)propanoate (2b) (89% yield)

Proton NMR (500 MHz, Chloroform-d) δ 8.11 (dd, J=2.8, 1.3 Hz, 1H), 7.56(dd, J=5.1, 1.3 Hz, 1H), 7.34 (dd, J=5.1, 2.8 Hz, 1H), 4.21 (q, J=7.1Hz, 2H), 3.89 (s, 2H), 1.25 (d, J=7.1 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃)δ 186.3, 167.2, 141.3, 133.3, 127.0, 126.7, 61.5, 47.2, 14.1. HRMS (ESI)Calcd for C₉H₁₁O₃S⁺ 199.0423 found 199.0431.

Ethyl 3-oxo-3-(pyridin-2-yl)propanoate (2c) (58% yield)

Proton NMR (500 MHz, Chloroform-d) δ 8.67 (ddd, J=4.8, 1.7, 0.9 Hz, 1H),8.08 (dt, J=8.0, 1.1 Hz, 1H), 7.86 (td, J=7.7, 1.7 Hz, 1H), 7.49 (ddd,J=7.6, 4.7, 1.3 Hz, 1H), 4.20 (m, 4H), 1.24 (t, J=7.1 Hz, 3H). ¹³C NMR(126 MHz, CDCl3) δ 194.9, 168.5, 152.6, 149.2, 137.2, 127.7, 122.3,61.4, 45.1, 14.3. HRMS (ESI) Calcd for C₁₀H₁₂NO₃ ⁺ 194.0812 found194.0809.

Ethyl 3-oxo-3-(pyridin-3-yl)propanoate (2d) (85% yield)

This compound is a mixture of ketone and enol forms (2/1). Proton NMR(500 MHz, Chloroform-d) (ketone) δ 9.16 (dd, J=2.3, 0.9 Hz, 1H), 8.82(dd, J=4.8, 1.7 Hz, 1H), 8.25 (ddd, J=8.0, 2.3, 1.7 Hz, 1H), 7.45 (ddd,J=8.0, 4.8, 0.9 Hz, 1H), 4.29 (q, J=7.1 Hz, 2H), 4.01 (s, 2H), 1.35 (t,J=7.1 Hz, 3H); (enol) δ 9.00 (dd, J=2.3, 0.9 Hz, 1H), 8.68 (dd, J=4.8,1.7 Hz, 1H), 8.06 (ddd, J=8.0, 2.3, 1.7 Hz, 1H), 7.37 (ddd, J=8.0, 4.8,0.9 Hz, 1H), 5.71 (s, 1H), 4.23 (q, J=7.2 Hz, 2H), 1.27 (t, J=7.1 Hz,3H). ¹³C NMR (126 MHz, CDCl₃ list of carbons for both the ketone and theenol) δ 191.4, 173.0, 169.0, 167.0, 154.0, 151.8, 150.0, 147.4, 135.8,133.4, 131.4, 129.4, 123.8, 123.4, 88.7, 61.8, 60.6, 46.0, 14.3, 14.1.

Ethyl 3-(4-chlorophenyl)-3-oxopropanoate (2e) 83% yield

This compound is a mixture of ketone and enol forms (2/1). Proton NMR(500 MHz, CDCl₃) (ketone) δ 1.24 (t, J=7.0 Hz, 3H), 3.95 (s, 2H), 4.20(q, J=7.0 Hz, 2H), 7.43 (d, J=7.0 Hz, 2H), 7.87 (d, J=7.0 Hz, 2H);(enol) δ 1.32 (t, J=7.5 Hz, 3H), 4.20 (q, J=7.5 Hz, 2H), 5.62 (s, 1H),7.37 (d, J=7.0 Hz, 2H), 7.69 (d, J=7.0 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃)(ketone) δ 13.0, 44.8, 60.4, 128.0, 128.9, 133.3, 139.1, 166.2, 190.3;(enol) δ 13.2, 59.4, 86.6, 126.3, 127.7, 130.8, 136.2, 168.9, 172.0.

Ethyl 3-(1-methyl-1H-pyrrol-3-yl)-3-oxopropanoate (2f) (62% yield)

Proton NMR (500 MHz, Chloroform-d) δ 7.27 (m, 1H), 6.57 (dt, J=2.1, 1.2Hz, 2H), 4.18 (q, J=7.1 Hz, 2H), 3.72 (s, 2H), 3.68 (s, 3H), 1.25 (t,J=7.1 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 187.0, 168.2, 127.6, 125.2,123.8, 110.0, 61.4, 47.2, 36.9, 14.3.

Ethyl 3-(2,5-dimethylthiophen-3-yl)-3-oxopropanoate (2g) (99% yield)

Proton NMR (500 MHz, Chloroform-d) δ 6.93 (q, J=1.2 Hz, 1H), 4.20 (q,J=7.1 Hz, 2H), 3.79 (s, 2H), 2.66 (s, 3H), 2.39 (s, 3H), 1.26 (t, J=7.1Hz, 3H). ¹³C NMR (126 MHz, CDCl3) δ 187.9, 167.6, 149.3, 135.5, 134.4,125.9, 61.3, 48.7, 16.1, 15.0, 14.1.

General Method for the Preparation of Pyrazolones

Hydrazine monohydrate 64% (2.25 ml, 29.4 mmol, 1.05 equiv.) was addeddropwise to a solution of the β-ketoester in absolute ethanol (28 mmol,c=0.19 M). The resulting orange solution was stirred under reflux for 3hours. Conversion was checked by NMR, (few drops of the reaction mixturewere dried under a stream of air and dissolved in CDCl₃) and upondisappearance of the starting material, the solvent was evaporated underreduced pressure and the resulting solid was triturated with acetone orethanol.

5-(Thiophen-2-yl)-2,4-dihydro-3H-pyrazol-3-one (4a) (43% yield)triturated with acetone. Proton NMR (500 MHz, DMSO-d6) δ 12.06 (bs,0.3H), 10.67 (bs, 0.7H), 9.66 (bs, 1H), 7.44 (bs, 1H), 7.32 (bs, 1H),7.07 (bs, 1H), 5.68 (s, 1H proton at C-4).

5-(Thiophen-3-yl)-2,4-dihydro-3H-pyrazol-3-one (4b) (24% yield),triturated with ethanol. Proton NMR (500 MHz, DMSO-d6) δ 7.69 (d, J=1.8Hz, 1H) 7.57 (dd, J=5.0, 2.9 Hz, 1H), 7.40 (dd, J=5.2, 1.3 Hz, 1H), 6.0(bs, 1H), 5.73 (s, 2H). ¹³C NMR (126 MHz, DMSO) δ 160.9, 139.7, 132.4,126.8, 125.5, 120.2, 86.7.

5-(Pyridin-2-yl)-2,4-dihydro-3H-pyrazol-3-one (4c) (54% yield),triturated with ethanol Proton NMR (500 MHz, DMSO-d6) δ 12.24 (bs, 1H),9.70 (bs, 1H), 8.54 (d, J=4.8 Hz, 1H), 7.78 (m, 2H), 7.28 (m, 1H), 6.03(s, 1H). ¹³C NMR (200 MHz, DMSO) δ 161.4, 149.2, 148.6, 142.5, 137.0,122.7, 119.4, 88.1.

5-(Pyridin-3-yl)-2,4-dihydro-3H-pyrazol-3-one (4d) (81% yield), solidwashed with ethanol after filtration. Proton NMR (500 MHz, DMSO-d6) δ8.87 (dd, J=2.3, 0.9 Hz, 1H), 8.44 (dd, J=4.8, 1.6 Hz, 1H), 8.00 (ddd,J=8.0, 2.3, 1.6 Hz, 1H), 7.60 (bs, 2H), 7.38 (ddd, J=7.9, 4.8, 0.9 Hz,1H), 5.86 (s, 1H). ¹³C NMR (126 MHz, DMSO) δ 161.1, 148.7, 146.4, 142.8,132.2, 128.3, 124.2, 86.2.

5-(4-Chlorophenyl)-2,4-dihydro-3H-pyrazol-3-one (4e) (27% yield),triturated with ethanol

5-(1-Methyl-1H-pyrrol-3-yl)-2,4-dihydro-3H-pyrazol-3-one (4f) (68%yield), triturated with ethanol. Proton NMR (500 MHz, DMSO-d6) δ 10.87(s, 1H), 9.50 (s, 1H), 6.97 (dd, J=1.9, 1.9 Hz, 1H), 6.67 (dd, J=2.4,2.4 Hz, 1H), 6.23 (dd, J=2.7, 1.7 Hz, 1H), 5.43 (s, 1H), 3.58 (s, 3H).¹³C NMR (126 MHz, DMSO) δ 161.3, 139.5, 122.5, 118.9, 114.1, 105.6,85.1, 35.7.

5-(2,5-Dimethylthiophen-3-yl)-2,4-dihydro-3H-pyrazol-3-one (4g) (32%yield), Proton NMR (500 MHz, DMSO-d6) δ 11.62 (s, 1H), 9.66 (s, 1H),6.91 (d, J=1.3 Hz, 1H), 5.59 (s, 1H), 2.41 (s, 3H), 2.36 (s, 3H). ¹³CNMR (126 MHz, DMSO) δ 161.1, 138.4, 135.0, 131.8, 127.4, 125.2, 88.1,14.7, 14.2.

2-Methyl-5-(thiophen-2-yl)-2,4-dihydro-3H-pyrazol-3-one (4h) (yield51%), triturated with ethanol. Proton NMR (500 MHz, DMSO-d6) δ 11.08 (s,1H), 7.34 (dd, J=5.0, 1.2 Hz, 1H), 7.24 (dd, J=3.5, 1.2 Hz, 1H), 7.00(dd, J=5.1, 3.5 Hz, 1H), 5.66 (s, 1H), 3.50 (s, 3H). ¹³C NMR (126 MHz,DMSO) δ 152.9, 143.4, 137.8, 127.3, 124.1, 123.0, 83.1, 33.1.

General Method for the Preparation of 2,4-dihydropyrano[2,3-c]pyrazoles

Pyrazolone (12 mmol, 1.1 equiv.) was suspended in absolute ethanol(typical concentration c=0.3 mM) and malononitrile (1.1 equiv.),aldehyde (1 equiv.), and triethyl amine (0.11 equiv.) were addedsequentially. The resulting suspension was stirred at room temperature(or at the temperature indicated) for 2 h, unless noted otherwise.Generally, the suspension clarified (due to the formation of polarintermediates), and as the reaction progressed, the productprecipitated. The solid was isolated by filtration under vacuum andwashed with cold ethanol.

6-Amino-4-(3-bromo-4-methoxyphenyl)-3-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(6a)

The title compound was isolated as colorless solid in quantitativeyield, starting from 3-bromo-4-methoxybenzaldehyde and 4a. Proton NMR(800 MHz, DMSO-d6) δ 13.03 (s, 1H), 7.58 (d, J=5.0 Hz, 1H), 7.27 (m,2H), 7.13 (dd, J=8.5, 1.8 Hz, 1H), 7.06 (m, 1H), 7.02 (d, J=8.5 Hz, 1H),6.98 (s, 2H), 4.79 (s, 1H), 3.80 (s, 3H). ¹³C NMR (200 MHz, DMSO) δ159.9, 155.9, 154.1, 138.0, 133.0, 131.6, 129.5, 128.0, 127.6, 127.4,125.9, 120.4, 112.6, 110.4, 96.8, 58.1, 56.1, 35.3. HRMS (ESI⁺) Calcdfor C₁₈H₁₄BrN₄O₂S⁺ [M+H]⁺ 429.0015 (100.0%), 430.9995 (97.3%), found429.0070 and 431.0051.

6-Amino-4-(4-methoxyphenyl)-3-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(6b)

The title compound was isolated as colorless solid in 64% yield,starting from 4-methoxybenzaldehyde and 4a. Proton NMR (800 MHz,DMSO-d6) δ 12.97 (s, 1H), 7.55 (dd, J=5.0, 1.2 Hz, 1H), 7.22 (dd, J=3.5,1.3 Hz, 1H), 7.04 (m, 3H), 6.90 (m, 2H), 6.83 (m, 2H), 4.72 (s, 1H),3.70 (s, 3H). ¹³C NMR (200 MHz, DMSO) δ 159.7, 158.0, 156.0, 136.4,132.8, 129.8, 128.5, 127.5, 127.1, 125.5, 120.6, 113.7, 97.3, 58.7,54.9, 35.8. HRMS (ESI⁺) Calcd for C₁₈H₁₅N₄O₂S⁺ [M+H]⁺ 351.0910 found351.0920.

6-Amino-4-(3-bromophenyl)-3-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(6c)

The title compound was isolated as colorless solid in 36% yield,starting from 3-bromobenzaldehyde and 4a. Proton NMR (800 MHz, DMSO-d6)δ 13.05 (s, 1H), 7.58 (m, 1H), 7.39 (ddd, J=8.0, 2.0, 1.0 Hz, 1H), 7.28(t, J=1.9 Hz, 1H), 7.26 (m, 2H), 7.15 (d, J=7.7 Hz, 1H), 7.05 (m, 3H),4.85 (s, 1H). ¹³C NMR (126 MHz, DMSO) δ 160.1, 155.8, 147.0, 133.0,130.7, 130.0, 130.0, 129.4, 127.6, 127.4, 126.6, 125.9, 121.6, 120.3,96.4, 57.6, 36.0. HRMS (ESI⁺) Calcd for C₁₇H₁₂BrN₄OS⁺ [M+H]⁺ 398.9910found 398.9914.

6-Amino-4-(4-hydroxyphenyl)-3-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(6d)

The title compound was isolated as colorless solid in 32% yield,starting from 4-hydroxybenzaldehyde and 4a. Proton NMR (800 MHz,DMSO-d6) δ 12.94 (s, 1H), 9.26 (s, 1H), 7.55 (dd, J=5.1, 1.1 Hz, 1H),7.20 (dd, J=3.6, 1.2 Hz, 1H), 7.04 (dd, J=5.1, 3.6 Hz, 1H), 6.92 (m,2H), 6.86 (s, 2H), 6.65 (m, 2H), 4.64 (s, 1H). ¹³C NMR (200 MHz, DMSO) δ160.2, 156.5, 135.2, 133.3, 130.3, 128.9, 128.0, 127.6, 126.0, 121.1,115.6, 98.0, 59.4, 49.1, 36.4. HRMS (ESI⁺) Calcd for C₁₇H₁₃N₄O₂S⁺ [M+H]⁺337.0754 found 337.0785.

6-Amino-3-(thiophen-2-yl)-4-(p-tolyl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(6e)

The title compound was isolated as colorless solid in 12% yield,starting from 4-methylbenzaldehyde and 4a. Proton NMR (500 MHz, DMSO-d6)δ 12.95 (s, 1H), 7.52 (d, J=5.0 Hz, 1H), 7.19 (d, J=3.6 Hz, 1H), 7.05(d, J=7.8 Hz, 2H), 7.03-6.94 (m, 3H), 6.88 (s, 2H), 4.69 (s, 1H), 2.21(s, 3H). ¹³C NMR (200 MHz, MeOD) δ 160.5, 156.4, 141.0, 136.4, 134.1,129.6, 128.8, 127.3, 127.0, 126.4, 125.8, 120.3, 97.7, 59.1, 36.7, 19.7.HRMS (ESI⁺) Calcd for C₁₈H₁₅N₄OS⁺ [M+H]⁺ 335.0961 found 335.0994.

6-Amino-4-(4-chlorophenyl)-3-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(6f)

The title compound was isolated as colorless solid in 21% yield,starting from 4-chlorobenzaldehyde and 4a. Proton NMR (500 MHz, DMSO-d6)δ 13.00 (s, 1H), 7.53 (dd, J=5.0, 1.2 Hz, 1H), 7.39-7.24 (m, 2H), 7.21(dd, J=3.7, 1.2 Hz, 1H), 7.16-7.04 (m, 2H), 7.02 (dd, J=5.1, 3.6 Hz,1H), 6.98 (s, 2H), 4.80 (s, 1H). ¹³C NMR (200 MHz, MeOD) δ 160.6, 156.2,142.7, 134.2, 132.4, 129.3, 129.0, 128.2, 127.1, 126.6, 126.0, 120.1,97.1, 58.3, 36.5. HRMS (ESI⁺) Calcd for C₁₇H₁₂ClN₄OS⁺ [M+H]⁺ 355.0415found 355.0414.

6-Amino-4-(4-nitrophenyl)-3-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(6g)

The title compound was isolated as colorless solid in 8% yield, startingfrom 4-nitrobenzaldehyde and 4a. Proton NMR (500 MHz, DMSO-d6) δ 13.08(s, 1H), 8.24-8.04 (m, 2H), 7.52 (dd, J=5.0, 1.2 Hz, 1H), 7.47-7.33 (m,2H), 7.24 (dd, J=3.7, 1.2 Hz, 1H), 7.10 (s, 2H), 7.02 (dd, J=5.1, 3.6Hz, 1H), 5.01 (s, 1H). ¹³C NMR (200 MHz, MeOD) δ 160.9, 156.2, 151.2,147.0, 134.3, 129.1, 128.6, 127.2, 126.7, 126.1, 123.4, 119.8, 96.5,57.3, 36.8. HRMS (ESI⁺) Calcd for C₁₇H₁₂N₅O₃S⁺ [M+H]⁺ 366.0655 found366.06821.

6-amino-4-(9H-fluoren-3-yl)-3-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(6h)

The title compound was isolated as colorless solid in 64% yield,starting from fluorene-2-carboxaldehyde and 4a. The reaction was rununder reflux. Proton NMR (800 MHz, DMSO-d6) δ 13.01 (s, 1H), 7.84 (d,J=7.6 Hz, 1H), 7.80 (d, J=7.8 Hz, 1H), 7.54 (d, J=7.5 Hz, 1H), 7.51 (d,J=5.0 Hz, 1H), 7.36 (t, J=7.4 Hz, 1H), 7.31-7.27 (m, 2H), 7.27-7.21 (m,2H), 7.02 (dd, J=5.0, 3.5 Hz, 1H), 6.97 (s, 2H), 4.87 (s, 1H), 3.88 (d,J=22.1 Hz, 1H), 3.83 (d, J=22.1 Hz, 1H). ¹³C NMR (200 MHz, DMSO) δ160.4, 156.5, 143.8, 143.7, 143.5, 141.2, 140.4, 133.5, 130.2, 128.0,127.7, 127.2, 127.1, 127.0, 126.2, 125.5, 124.4, 121.0, 120.3, 120.2,97.8, 59.0, 37.2, 36.8. HRMS (ESI⁺) Calcd for C₂₄H₁₇N₄OS⁺=409.1118,found 409.1121.

4-(1-Allylindolin-6-yl)-6-amino-3-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(6i)

The title compound was isolated as a pale yellow solid in 34% yield,starting from 1-allyl-2,3-dihydro-1H-indole-5-carbaldehyde and 4a. Thereaction was run at 60° C. for 3h. Proton NMR (800 MHz, DMSO-d6) δ 12.92(s, 1H), 7.56 (d, J=5.1 Hz, 1H), 7.24 (d, J=3.5 Hz, 1H), 7.05 (dd,J=5.1, 3.5 Hz, 1H), 6.83 (m, 3H), 6.72 (d, J=1.8 Hz, 1H), 6.42 (d, J=8.0Hz, 1H), 5.86 (ddt, J=17.2, 10.2, 6.0 Hz, 1H), 5.27 (dq, J=17.2, 1.7 Hz,1H), 5.17 (dq, J=10.2, 1.4 Hz, 1H), 4.60 (s, 1H), 3.66 (ddt, J=5.8, 4.3,1.5 Hz, 2H), 3.24 (t, J=8.3 Hz, 2H), 2.80 (ddd, J=32.5, 15.7, 7.6 Hz,2H). ¹³C NMR (200 MHz, DMSO-d6) δ 160.2, 156.5, 151.4, 134.8, 134.0,133.2, 130.7, 130.4, 128.0, 127.6, 126.9, 125.9, 123.5, 121.2, 117.7,107.0, 98.4, 59.8, 53.1, 51.8, 36.7, 28.3. HRMS (ESI⁺) Calcd forC₂₂H₂₀N₅OS⁺=402.1384, found 402.1386.

6-amino-4-(4-(benzyloxy)-3,5-dibromophenyl)-3-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(61)

The title compound was isolated as colorless solid in 69% yield,starting from 4-benzyloxy-3,5-dibromo-benzaldehyde and 4a. The reactionwas run at 60° C. for 1 h. Proton NMR (800 MHz, DMSO-d6) δ 13.09 (s,1H), 7.63 (dd, J=5.1, 1.2 Hz, 1H), 7.52 (m, 2H), 7.48-7.33 (m, 5H), 7.31(dd, J=3.6, 1.2 Hz, 1H), 7.10 (m, 3H), 4.96 (s, 2H), 4.92 (s, 1H). ¹³CNMR (200 MHz, DMSO) δ 160.8, 156.2, 151.2, 144.1, 136.5, 133.6, 132.1,129.7, 128.9, 128.8, 128.8 (overlapping), 128.7, 128.1, 128.1, 126.8,120.7, 118.3, 96.6, 74.8, 57.5, 55.4, 35.6. HRMS (ESI⁺) Calcd forC₂₄H₁₇Br₂N₄O₂S⁺=584.9414, found 584.9413.

6-Amino-4-(3-bromo-4-propoxyphenyl)-3-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(6m)

The title compound was isolated as colorless solid in 64% yield,starting from 3-bromo-4-propoxybenzaldehyde and 4a. The reaction was runat 60° C. for 2h. Proton NMR (800 MHz, DMSO-d6) δ 12.99 (s, 1H), 7.56(dt, J=5.1, 1.0 Hz, 1H), 7.25 (m, 2H), 7.07 (dd, J=8.5, 2.2 Hz, 1H),7.05 (ddd, J=4.7, 3.8, 0.8 Hz, 1H), 6.98 (d, J=8.5 Hz, 1H), 6.94 (s,2H), 4.76 (s, 1H), 3.94 (t, J=6.3 Hz, 2H), 1.71 (dq, J=7.7, 6.6 Hz, 2H),0.98 (td, J=7.4, 0.8 Hz, 3H). ¹³C NMR (200 MHz, DMSO-d6) δ 159.9, 155.8,153.5, 137.9, 133.0, 131.5, 129.5, 127.9, 127.6, 127.3, 125.8, 120.3,113.5, 110.8, 96.8, 70.0, 58.1, 35.3, 22.0, 10.4. HRMS (ESP) Calcd forC₂₀H₁₈BrN₄O₂S⁺=457.0328, found 457.0328.

6-Amino-4-(3-bromo-4-(prop-2-yn-1-yloxy)phenyl)-3-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(6n)

The title compound was isolated as off-white solid in 55% yield,starting from 3-bromo-4-(2-propyn-1-yloxy)benzaldehyde and 4a. Thereaction was run at 60° C. for 1 h. Proton NMR (800 MHz, DMSO-d6) δ13.02 (s, 1H), 7.58 (dd, J=5.1, 1.2 Hz, 1H), 7.29 (d, J=2.2 Hz, 1H),7.26 (dd, J=3.6, 1.2 Hz, 1H), 7.13 (dd, J=8.6, 2.2 Hz, 1H), 7.09 (d,J=8.6 Hz, 1H), 7.06 (dd, J=5.1, 3.6 Hz, 1H), 6.98 (s, 2H), 4.86 (d,J=2.4 Hz, 2H), 4.80 (s, 1H), 3.59 (t, J=2.3 Hz, 1H). ¹³C NMR (200 MHz,DMSO-d6) δ 160.5, 156.3, 152.7, 139.3, 133.5, 132.2, 130.0, 128.3,128.1, 127.8, 126.4, 120.9, 114.6, 111.3, 97.2, 79.3, 79.2, 58.5, 56.9,35.8. HRMS (ESI⁺) Calcd for C₂₀H₁₄BrN₄O₂S⁺=453.0016, found 453.0013.

6-Amino-4-(2,3-dihydrobenzofuran-5-yl)-3-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(6o)

The title compound was isolated as colorless solid in 60% yield,starting from 2,3-dihydrobenzofuran-5-carbaldehyde and 4a. The reactionwas run at room temperature for 12 h. Proton NMR (800 MHz, DMSO-d6) δ12.96 (s, 1H), 7.56 (dd, J=5.1, 1.2 Hz, 1H), 7.24 (dd, J=3.7, 1.2 Hz,1H), 7.05 (dd, J=5.1, 3.6 Hz, 1H), 6.90 (m, 4H), 6.65 (d, J=8.1 Hz, 1H),4.69 (s, 1H), 4.46 (m, 2H), 3.10 (m, 2H). ¹³C NMR (126 MHz, DMSO) δ169.0, 159.8, 158.5, 156.0, 136.5, 132.8, 129.8, 127.5, 127.2, 125.6,123.8, 120.6, 109.2, 108.3, 97.6, 70.9, 58.9, 36.1, 29.0. HRMS (ESI⁺)Calcd for C₁₉H₁₅N₄O₂S⁺ [M+H]⁺ 363.0910 found 363.0941.

6-Amino-4-(4-azido-3-iodophenyl)-3-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(6p)

The title compound was isolated as colorless solid in 24% yield,starting from 4-azido-3-iodobenzaldehyde and 4a. The reaction was run atroom temperature for 12 h. Proton NMR (800 MHz, DMSO-d6) δ 13.04 (s,1H), 7.59 (dd, J=5.1, 1.2 Hz, 1H), 7.56 (d, J=2.0 Hz, 1H), 7.28 (dd,J=3.6, 1.2 Hz, 1H), 7.25 (d, J=8.3 Hz, 1H), 7.22 (dd, J=8.3, 2.0 Hz,1H), 7.07 (dd, J=5.0, 3.6 Hz, 1H), 7.02 (s, 2H), 4.83 (s, 1H). ¹³C NMR(200 MHz, DMSO) δ 160.0, 155.7, 142.8, 139.6, 138.2, 133.0, 129.3,129.0, 127.6, 127.4, 126.0, 120.3, 119.4, 96.4, 57.6, 48.6, 35.2. HRMS(ESI⁺) Calcd for C₁₇H₁₁IN₇OS⁺ [M+H]⁺ 487.9785 found 487.9806.

6-Amino-4-(4-hydroxy-3-(trifluoromethyl)phenyl)-3-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(6q)

The title compound was isolated as colorless solid in 14% yield,starting from 4-hydroxy-3-trifluoromethylbenzaldehyde and 4a. Thereaction was run at room temperature for 12 h. Proton NMR (500 MHz,DMSO-d6) δ 12.97 (s, 1H) 10.42 (s, 1H), 7.54 (dd, J=5.1, 1.2 Hz, 1H),7.22 (dt, J=4.9, 1.7 Hz, 2H), 7.15 (dd, J=8.5, 2.3 Hz, 1H), 7.02 (dd,J=5.1, 3.6 Hz, 1H), 6.94 (s, 2H), 6.89 (d, J=8.5 Hz, 1H), 4.79 (s, 1H).¹³C NMR (126 MHz, DMSO) δ 159.9, 155.8, 154.5, 134.6, 133.0, 132.7,129.4, 127.5, 127.3, 125.9, 125.3, 125.0, 120.4, 117.2, 114.81 (q,J=30.0, 29.5 Hz), 96.9, 58.0, 35.5. HRMS (ESI⁺) Calcd for C₁₈H₁₂F₃N₄O₂S⁺[M+H]⁺ 405.0628 found 405.0626.

6-Amino-4-(5-isopropyl-4-methoxy-2-methylphenyl)-3-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(6r)

The title compound was isolated as colorless solid in 71% yield,starting from 5-isopropyl-4-methoxy-2-methylbenzaldehyde and 4a. Thereaction was run at 60° C. for 1 h. Proton NMR (800 MHz, DMSO-d6) δ12.86 (s, 1H), 7.52 (d, J=4.9 Hz, 1H), 7.09 (d, J=3.4 Hz, 1H), 7.00 (dd,J=5.0, 3.6 Hz, 1H), 6.82 (s, 2H), 6.75 (s, 1H), 6.67 (s, 1H), 4.95 (s,1H), 3.75 (s, 3H), 3.07 (hept, J=6.9 Hz, 1H), 2.28 (s, 3H), 1.05 (d,J=6.9 Hz, 3H), 0.97 (d, J=6.9 Hz, 3H). ¹³C NMR (200 MHz, DMSO) δ 160.4,156.6, 155.2, 134.0, 133.9, 133.9 (overlapping), 133.6, 133.2, 130.3,127.8, 127.7, 126.1, 121.1, 113.3, 98.1, 58.2, 55.7, 40.5, 26.4, 23.1,22.9, 19.3. HRMS (ESI⁺) Calcd for C₂₂H₂₃N₄O₂S⁺=407.1537, found 407.1539.

6-Amino-4-(3-fluoro-4-methoxyphenyl)-3-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(6s)

The title compound was isolated as colorless solid in quantitativeyield, starting from 3-fluoro-4-methoxybenzaldehyde and 4a. Proton NMR(500 MHz, DMSO-d6) δ 12.98 (s, 1H), 7.55 (dd, J=5.0, 1.2 Hz, 1H), 7.24(dd, J=3.6, 1.2 Hz, 1H), 7.08-7.01 (m, 2H), 6.98-6.87 (m, 4H), 4.76 (s,1H), 3.77 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 159.9, 155.9, 151.1 (d,J=244.3 Hz), 145.8 (d, J=10.5 Hz), 137.3 (d, J=4.6 Hz), 133.0, 129.6,127.6, 127.3, 125.8, 123.5, 120.4, 114.8 (d, J=18.0 Hz), 113.7, 96.7,58.0, 55.9, 35.6. HRMS (ESI⁺) Calcd for C₁₈H₁₄FN₄O₂S⁺=369.0816, found.369.0811.

6-Amino-4-(3-chloro-4-methoxyphenyl)-3-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(6t)

The title compound was isolated as colorless solid in 42% yield,starting from 3-chloro-4-methoxybenzaldehyde and 4a. The reaction wasrun under reflux. Proton NMR (800 MHz, DMSO-d6) δ 13.01 (s, 1H), 7.57(d, J=5.0 Hz, 1H), 7.27 (d, J=3.6 Hz, 1H), 7.09 (m, 4H), 6.96 (s, 2H),4.79 (s, 1H), 3.81 (s, 3H). ¹³C NMR (200 MHz, DMSO) δ 160.4, 156.3,153.7, 138.1, 133.5, 130.0, 129.0, 128.1, 127.8, 127.8, 126.3, 121.2,120.8, 113.2, 97.2, 58.6, 56.5, 35.9. HRMS (ESI⁺) Calcd forC₁₈H₁₄ClN₄O₂S⁺=385.0521, found 385.0543.

6-Amino-4-(3-iodo-4-methoxyphenyl)-3-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(6u)

The title compound was isolated as colorless solid in 60% yield,starting from 3-iodo-4-methoxybenzaldehyde and 4a. The reaction was runat room temperature overnight. Proton NMR (800 MHz, DMSO-d6) δ 13.00 (s,1H), 7.58 (dd, J=5.0, 1.2 Hz, 1H), 7.46 (d, J=2.2 Hz, 1H), 7.26 (dd,J=3.7, 1.2 Hz, 1H), 7.14 (dd, J=8.5, 2.2 Hz, 1H), 7.06 (dd, J=5.0, 3.7Hz, 1H), 6.95 (s, 2H), 6.91 (d, J=8.5 Hz, 1H), 4.76 (s, 1H), 3.78 (s,3H). ¹³C NMR (200 MHz, CDCl3) δ 159.9, 156.5, 155.8, 138.5, 137.6,133.0, 129.5, 128.8, 127.6, 127.4, 125.8, 120.4, 111.4, 96.9, 86.0,58.2, 56.3, 35.1. HRMS (ESI⁺) Calcd for C₁₈H₁₄IN₄O₂S⁺=476.9877, found476.9860.

6-Amino-4-(4-hydroxy-3-(trifluoromethyl)phenyl)-3-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(6v)

The title compound was isolated as colorless solid in 29% yield,starting from 4-methoxy-3-(trifluoromethyl)benzaldehyde and 4a. Thereaction was run at 60° C. Proton NMR (500 MHz, DMSO-d6) δ 13.00 (s,1H), 7.54 (dd, J=5.0, 1.2 Hz, 1H), 7.38-7.28 (m, 2H), 7.24 (dd, J=3.6,1.2 Hz, 1H), 7.14 (d, J=8.6 Hz, 1H), 7.02 (dd, J=5.1, 3.6 Hz, 1H), 6.98(s, 2H), 4.87 (s, 1H), 3.81 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 160.4,156.3, 136.7, 133.5, 130.9, 129.8, 128.1, 127.9, 126.5, 125.9, 125.2,123.1, 120.8, 116.9, 113.5, 97.1, 58.3, 56.5, 35.8. HRMS (ESI⁺) Calcdfor C₁₉H₁₄F₃N₄O₂S⁺=419.0785, found 419.0789.

6-Amino-4-(3-bromo-4-methoxyphenyl)-3-(thiophen-3-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(7a)

The title compound was isolated as colorless solid in 50% yield,starting from 3-bromo-4-methoxybenzaldehyde and 4b. The reaction was runat room temperature overnight. Proton NMR (500 MHz, DMSO-d6) δ 12.85 (s,1H), 7.57 (m, 2H), 7.29 (m, 2H), 7.14 (dd, J=8.5, 2.2 Hz, 1H), 6.99 (d,J=8.5 Hz, 1H), 6.95 (m, 2H), 4.94 (s, 1H), 3.77 (s, 3H). ¹³C NMR (126MHz, DMSO) δ 160.0, 155.7, 154.0, 138.4, 134.2, 131.4, 129.1, 127.8,127.0, 125.5, 123.0, 120.5, 112.5, 110.3, 96.5, 58.0, 56.1, 35.1. HRMS(ESI⁺) Calcd for C₁₈H₁₄BrN₄O₂S⁺=429.0016, found 429.0009.

6-Amino-4-(3-iodo-4-methoxyphenyl)-3-(thiophen-3-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(7b)

The title compound was isolated as colorless solid in 59% yield,starting from 3-iodo-4-methoxybenzaldehyde and 4b. The reaction was runat room temperature for 3h. Proton NMR (500 MHz, DMSO-d6) δ 12.83 (s,1H), 7.55 (m, 2H), 7.48 (d, J=2.2 Hz, 1H), 7.27 (dd, J=4.9, 1.5 Hz, 1H),7.14 (dd, J=8.5, 2.2 Hz, 1H), 6.92 (s, 2H), 6.86 (d, J=8.5 Hz, 1H), 4.89(s, 1H), 3.73 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 160.0, 156.3, 155.7,138.8, 137.5, 134.2, 129.1, 128.7, 127.0, 125.5, 123.0, 120.5, 111.4,96.6, 86.0, 58.1, 56.2, 34.9. HRMS (ESI⁺) Calcd forC₁₈H₁₄IN₄O₂S⁺=476.9877, found 476.9870.

6-Amino-4-(3-bromo-4-methoxyphenyl)-3-(pyridin-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(7c)

The title compound was isolated as colorless solid in 86% yield,starting from 3-bromo-4-methoxybenzaldehyde and 4c. The reaction was runat room temperature overnight. Proton NMR (500 MHz, DMSO-d6) δ 13.20 (s,1H), 8.54 (ddd, J=4.9, 1.9, 0.9 Hz, 1H), 7.75 (td, J=7.8, 1.8 Hz, 1H),7.55 (dt, J=8.0, 1.1 Hz, 1H), 7.30 (d, J=2.2 Hz, 1H), 7.27 (ddd, J=7.6,4.8, 1.0 Hz, 1H), 7.12 (dd, J=8.5, 2.2 Hz, 1H), 6.98 (s, 2H), 6.93 (d,J=8.6 Hz, 1H), 5.04 (s, 1H), 3.73 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ160.6, 156.30, 154.3, 149.8, 147.50, 139.1, 137.4, 132.1, 128.4, 125.36,123.6, 121.0, 120.9, 112.8, 110.5, 99.4, 58.4, 56.5, 36.3. HRMS (ESI⁺)Calcd for C₁₉H₁₅BrN₅O₂ ⁺=424.0404 found 424.0401.

6-Amino-4-(3-iodo-4-methoxyphenyl)-3-(pyridin-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(7d)

The title compound was isolated as colorless solid in 77% yield,starting from 3-iodo-4-methoxybenzaldehyde and 4c. The reaction was runat room temperature overnight. Proton NMR (500 MHz, DMSO-d6) δ 13.19 (s,1H), 8.54 (ddd, J=4.9, 1.8, 0.9 Hz, 1H), 7.76 (td, J=7.8, 1.8 Hz, 1H),7.57 (dt, J=8.1, 1.0 Hz, 1H), 7.52 (s, 1H), 7.29 (ddd, J=7.6, 4.8, 1.1Hz, 1H), 7.16 (dd, J=8.5, 2.2 Hz, 1H), 6.99 (s, 2H), 6.84 (d, J=8.5 Hz,1H), 5.03 (s, 1H), 3.72 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 160.2, 156.2,155.8, 149.3, 147.0, 139.1, 137.8, 136.9, 128.8, 123.1, 120.6, 120.5,111.2, 98.9, 85.6, 58.0, 56.2, 35.7. HRMS (ESI⁺) Calcd for C₁₉H₁₅IN₅O₂⁺=472.0265, found 472.0266.

6-Amino-4-(3-bromo-4-methoxyphenyl)-3-(pyridin-3-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(7e)

The title compound was isolated as colorless solid in 38% yield,starting from 3-bromo-4-methoxybenzaldehyde and 4d. The reaction was runat room temperature. Proton NMR (500 MHz, DMSO-d6) δ 13.10 (s, 1H), 8.65(dd, J=2.4, 0.9 Hz, 1H), 8.47 (dd, J=4.8, 1.6 Hz, 1H), 7.87 (ddd, J=8.0,2.4, 1.6 Hz, 1H), 7.37 (ddd, J=8.0, 4.8, 0.9 Hz, 1H), 7.27 (d, J=2.2 Hz,1H), 7.08 (dd, J=8.5, 2.3 Hz, 1H), 7.00 (s, 2H), 6.94 (d, J=8.5 Hz, 1H),5.09 (s, 1H), 3.75 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 160.0, 155.8,154.0, 149.2, 147.0, 137.9, 135.3, 133.7, 131.6, 127.9, 124.6, 123.5,120.4, 112.5, 110.2, 98.0, 57.8, 56.0, 35.1. HRMS (ESP) Calcd forC₁₉H₁₅BrN₅O₂ ⁺=424.0404, found 424.0403.

6-Amino-4-(3-iodo-4-methoxyphenyl)-3-(pyridin-3-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(7f)

The title compound was isolated as colorless solid in 5% yield, startingfrom 3-iodo-4-methoxybenzaldehyde and 4d. The reaction was run at roomtemperature. Proton NMR (500 MHz, DMSO-d6) δ 13.09 (s, 1H), 8.62 (dd,J=2.4, 0.9 Hz, 1H), 8.45 (dd, J=4.8, 1.6 Hz, 1H), 7.84 (dt, J=8.0, 1.8Hz, 1H), 7.43 (d, J=2.2 Hz, 1H), 7.34 (ddd, J=8.0, 4.9, 0.9 Hz, 1H),7.07 (dd, J=8.5, 2.3 Hz, 1H), 6.96 (s, 2H), 6.81 (d, J=8.5 Hz, 1H), 5.03(s, 1H), 3.70 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 160.0, 156.4, 155.8,149.2, 147.1, 138.4, 137.6, 135.3, 133.7, 128.7, 124.6, 123.5, 120.4,111.3, 98.2, 85.9, 57.9, 56.2, 35.0. HRMS (ESP) Calcd for C₁₉H₁₅IN₅O₂⁺=472.0265, found 472.0265.

6-Amino-3-(4-chlorophenyl)-4-(3,4-dimethylphenyl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(7g)

The title compound was isolated as colorless solid in 75% yield,starting from 3,4-dimethylbenzaldehyde and 4e. The reaction was run atroom temperature. Proton NMR (800 MHz, DMSO-d6) δ 12.90 (s, 1H), 7.48(m, 2H), 7.38 (m, 2H), 6.95 (d, J=8.3 Hz, 1H), 6.87 (s, 2H), 6.82 (d,J=6.4 Hz, 2H), 4.88 (s, 1H), 2.10 (s, 3H), 2.09 (s, 3H). ¹³C NMR (200MHz, DMSO) δ 159.9, 156.0, 141.9, 136.6, 136.0, 134.4, 133.0, 129.3,128.6, 128.2, 128.0, 127.5, 124.7, 120.5, 98.1, 58.4, 36.3, 19.4, 18.9.HRMS (ESI⁺) Calcd for C₂₁H₁₈ClN₄O⁺=377.1164, found 377.1164.

6-Amino-4-(3-bromo-4-methoxyphenyl)-3-(4-chlorophenyl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(7h)

The title compound was isolated as colorless solid in 49% yield,starting from 3-bromo-4-methoxybenzaldehyde and 4e. The reaction was runat room temperature. Proton NMR (800 MHz, DMSO-d6) δ 12.96 (s, 1H), 7.50(m, 2H), 7.39 (d, J=8.5 Hz, 2H), 7.23 (d, J=2.2 Hz, 1H), 7.08 (dd,J=8.5, 2.2 Hz, 1H), 6.94 (m, 3H), 5.01 (s, 1H), 3.75 (s, 3H). ¹³C NMR(200 MHz, DMSO) δ 160.0, 155.8, 154.0, 138.1, 136.9, 133.1, 131.5,128.6, 128.1, 127.8, 127.3, 120.4, 112.4, 110.2, 97.6, 57.9, 56.1, 35.3.HRMS (ESI⁺) Calcd for C₂₀H₁₈BrClN₄O₂ ⁺=457.0062, found 457.0073.

6-Amino-3-(4-chlorophenyl)-4-(3-iodo-4-methoxyphenyl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrilem(7i)

The title compound was isolated as colorless solid in 61% yield,starting from 3-iodo-4-methoxybenzaldehyde and 4e. The reaction was runat room temperature. Proton NMR (800 MHz, DMSO-d6) δ 12.98 (s, 1H), 7.51(m, 2H), 7.41 (m, 2H), 7.25 (d, J=2.2 Hz, 1H), 7.09 (dd, J=8.5, 2.2 Hz,1H), 6.96 (m, 3H), 5.03 (s, 1H), 3.76 (s, 3H). ¹³C NMR (200 MHz, DMSO) δ160.0, 155.8, 154.0, 138.1, 136.9, 133.1, 131.5, 128.6, 128.1, 127.8,127.3, 120.4, 112.4, 110.2, 97.6, 57.9, 56.1, 35.3. HRMS (ESI⁺) Calcdfor C₂₀H₁₅ClIN₄O₂ ⁺=504.9923, found 504.9938.

6-Amino-4-(3-bromo-4-methoxyphenyl)-3-(1-methyl-1H-pyrrol-3-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(71)

The title compound was isolated as colorless solid in 96% yield,starting from 3-bromo-4-methoxybenzaldehyde and 4f. The reaction was runat room temperature. Proton NMR (500 MHz, DMSO-d6) δ 12.35 (s, 1H), 7.27(d, J=2.1 Hz, 1H), 7.13 (dd, J=8.5, 2.1 Hz, 1H), 6.99 (d, J=8.6 Hz, 1H),6.84 (s, 2H), 6.79 (d, J=1.9 Hz, 1H), 6.61 (d, J=2.4 Hz, 1H), 6.12 (dd,J=2.8, 1.7 Hz, 1H), 4.69 (s, 1H), 3.77 (s, 3H), 3.50 (s, 3H). ¹³C NMR(126 MHz, DMSO) δ 160.1, 155.6, 153.9, 138.8, 135.2, 131.5, 127.9,122.6, 120.7, 120.2, 112.5, 111.7, 110.3, 105.9, 94.1, 58.2, 56.1, 35.8,35.3. HRMS (ESI⁺) Calcd for C₁₉H₁₇BrN₅O₂ ⁺=426.0560, found 426.0555.

6-Amino-4-(3-iodo-4-methoxyphenyl)-3-(1-methyl-1H-pyrrol-3-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(7m)

The title compound was isolated as colorless solid in 75% yield,starting from 3-iodo-4-methoxybenzaldehyde and 4f. The reaction was runat room temperature. Proton NMR (500 MHz, DMSO-d6) δ 12.34 (s, 1H), 7.48(d, J=2.1 Hz, 1H), 7.14 (dd, J=8.5, 2.2 Hz, 1H), 6.88 (d, J=8.5 Hz, 1H),6.84 (s, 2H), 6.79 (t, J=1.9 Hz, 1H), 6.61 (t, J=2.4 Hz, 1H), 6.12 (dd,J=2.7, 1.7 Hz, 1H), 4.65 (s, 1H), 3.75 (s, 3H), 3.51 (s, 3H). ¹³C NMR(126 MHz, DMSO) δ 160.1, 156.3, 155.6, 139.3, 137.5, 135.2, 128.7,122.5, 120.7, 120.2, 111.7, 111.3, 105.9, 94.3, 85.9, 58.3, 56.3, 35.8,35.2. HRMS (ESI⁺) Calcd for C₁₉H₁₇IN₅O₂ ⁺=474.0421, found 474.0424.

6-Amino-4-(3-bromo-4-methoxyphenyl)-3-(2,5-dimethylthiophen-3-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(7n)

The title compound was isolated as colorless solid in 49% yield,starting from 3-bromo-4-methoxybenzaldehyde and 4g. The reaction was runat room temperature. Proton NMR (500 MHz, DMSO-d6) δ 12.47 (s, 1H), 7.09(d, J=2.0 Hz, 1H), 7.03-6.91 (m, 4H), 6.31 (d, J=1.4 Hz, 1H), 4.64 (s,1H), 3.78 (s, 3H), 2.42-2.25 (m, 3H), 2.15 (s, 3H). ¹³C NMR (126 MHz,DMSO) δ 160.7, 154.7, 153.9, 138.2, 135.4, 135.2, 134.6, 131.6, 127.6,126.4, 125.6, 120.6, 112.3, 110.1, 98.4, 56.9, 56.1, 35.3, 14.6, 13.4.HRMS (ESI⁺) Calcd for C₂₀H₁₈BrN₄O₂S⁺=457.0328, found 457.0329.

6-Amino-3-(2,5-dimethylthiophen-3-yl)-4-(3-iodo-4-methoxyphenyl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(7o)

The title compound was isolated as colorless solid in 18% yield,starting from 3-iodo-4-methoxybenzaldehyde and 4g. The reaction was runat room temperature. Proton NMR (500 MHz, DMSO-d6) δ 12.43 (s, 1H), 7.27(d, J=2.2 Hz, 1H), 7.00-6.92 (m, 3H), 6.81 (d, J=8.5 Hz, 1H), 6.29 (d,J=1.3 Hz, 1H), 4.59 (s, 1H), 3.73 (s, 3H), 2.28 (s, 3H), 2.12 (s, 3H).¹³C NMR (126 MHz, DMSO) δ 160.7, 156.3, 154.7, 138.7, 137.7, 135.3,135.2, 134.6, 128.4, 126.4, 125.6, 120.6, 111.2, 98.5, 85.5, 56.9, 56.3,35.1, 14.7, 13.4. HRMS (ESI⁺) Calcd for C₂₀H₁₈IN₄O₂S⁺=505.0190, found505.0181.

Ethyl6-amino-4-(3-bromo-4-methoxyphenyl)-3-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carboxylate(8)

Ethyl cyanoacetate (20.0 μl, 0.185 mmol) and triethyl amine (26.0 μl,0.185 mmol) were added to a suspension of 3-Br-4-OMebenzaldehyde (38 mg,0.176 mmol), in 2 ml of ethanol. The reaction was stirred at roomtemperature for 4 h (white suspension) and complete formation of thearylidene malononitrile was verified by tlc. Pyrazolone 4a was added andthe reaction mixture was stirred at room temperature for 5 days. Theprecipitate was filtered under vacuum and the mother liquor wasconcentrated and purified by automated flash chromatography, using a 16g silica gel column and a mixture of hexanes and ethyl acetate. Thegradient was as follow: 10% ethyl acetate 3 min, 30% 10 min, 30% 15 min,50% 20 min, 50% 30 min, 100% 33 min, 100% 43 min. The title compound wasisolated in 15% yield as a colorless glassy solid. Proton ¹H NMR (500MHz, DMSO-d6) δ 12.89 (s, 1H), 7.78 (s, 2H), 7.60 (dd, J=5.1, 1.2 Hz,1H), 7.31 (dd, J=3.6, 1.2 Hz, 1H), 7.27 (d, J=2.2 Hz, 1H), 7.09 (dd,J=5.1, 3.6 Hz, 1H), 7.03 (dd, J=8.5, 2.2 Hz, 1H), 6.91 (d, J=8.6 Hz,1H), 4.90 (s, 1H), 4.03 (dq, J=10.9, 7.1 Hz, 1H), 3.96 (dq, J=10.9, 7.1Hz, 1H), 3.73 (s, 3H), 1.17 (t, J=7.1 Hz, 3H). ¹³C NMR (126 MHz, DMSO) δ168.3, 161.0, 156.8, 153.4, 140.1, 132.3, 129.8, 128.2, 127.7, 127.2,127.1, 125.3, 112.1, 109.4, 99.9, 77.5, 58.9, 56.0, 34.0, 14.2. HRMS(ESI⁺) Calcd for C₂₀H₁₉BrN₃O₄S⁺ [M+H]⁺ 476.0274 (100%), 478.0254 (97%),found 476.0274 (100%), 478.0256 (97%).

6-Amino-4-(3-bromo-4-methoxyphenyl)-1-methyl-3-(thiophen-2-yl)-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(9a)

The title compound was isolated as colorless solid in 64% yield,starting from 3-bromo-4-methoxybenzaldehyde and 4h. The reaction was runat room temperature for 3h. Proton NMR (500 MHz, DMSO-d6) δ 7.34 (dd,J=4.9, 1.3 Hz, 1H), 7.33 (d, J=2.2 Hz, 1H), 7.19 (dd, J=8.5, 2.2 Hz,1H), 7.12 (s, 2H), 7.00 (d, J=8.5 Hz, 1H), 6.89 (m, 2H), 4.85 (s, 1H),3.78 (s, 3H), 3.73 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 158.5, 154.1,145.6, 140.0, 137.7, 135.2, 131.6, 128.1, 127.5, 125.1, 124.7, 119.9,112.4, 110.5, 94.2, 59.2, 56.1, 36.0, 34.0. HRMS (ESI⁺) Calcd forC₁₉H₁₆BrN₄O₂S⁺=443.0172, found 443.0171.

6-Amino-4-(3-iodo-4-methoxyphenyl)-1-methyl-3-(thiophen-2-yl)-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(9b)

The title compound was isolated as colorless solid in 17% yield,starting from 3-iodo-4-methoxybenzaldehyde and 4h. The reaction was runat room temperature for 3h. Proton NMR (500 MHz, DMSO-d6) δ 7.52 (d,J=2.2 Hz, 1H), 7.36 (dd, J=4.9, 1.3 Hz, 1H), 7.22 (dd, J=8.5, 2.2 Hz,1H), 7.13 (s, 2H), 6.96-6.74 (m, 3H), 4.84 (s, 1H), 3.77 (s, 3H), 3.74(s, 3H). ¹³C NMR (126 MHz, DMSO) δ 158.5, 156.5, 145.6, 140.0, 138.2,137.6, 135.2, 129.0, 127.5, 125.1, 124.8, 120.0, 111.3, 94.3, 86.2,59.3, 56.3, 35.8, 34.0. HRMS (ESI⁺) Calcd for C₁₉H₁₆IN₄O₂S⁺=491.0033,found 491.0026.

4-(3-Bromo-4-methoxyphenyl)-6-((ethoxymethyl)amino)-1-methyl-3-(thiophen-2-yl)-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(11)

Compound 9a (50.0 mg, 0.11 mmol) was suspended in 0.80 ml of triethylorthoformate and 0.20 ml of glacial acetic acid were added dropwise. Thereaction mixture was heated to 110° C. in oil bath, in about 30 min andconversion was judged complete by tlc (R_(f)=0.45 using hexanes, ethylacetate 1:1). The mixture was concentrated to dryness, and the crude wassuspended in 1.00 ml of EtOH. Sodium borohydrate (6.4 mg, 0.17 mmol) wasadded and the reaction mixture was stirred at room temperature for 3h.Upon complete conversion (monitored by tlc, R_(f)=0.30 using hexanes,ethyl acetate 1:1), the title compound was isolated by filtration undervacuum and washed first with ethyl ether and then with few drops of coldethanol. The title compound was isolated as colorless solid in 62%yield. Proton NMR (500 MHz, DMSO-d6) δ 8.16 (t, J=6.6 Hz, 1H), 7.38 (dd,J=4.5, 1.8 Hz, 1H), 7.36 (d, J=2.2 Hz, 1H), 7.22 (dd, J=8.5, 2.2 Hz,1H), 7.03 (d, J=8.5 Hz, 1H), 6.93 (m, 2H), 4.96 (s, 1H), 4.76 (dd,J=10.8, 6.9 Hz, 1H), 4.61 (dd, J=10.8, 6.3 Hz, 1H), 3.80 (s, 3H), 3.78(s, 3H), 3.51 (qd, J=7.0, 4.5 Hz, 2H), 1.12 (t, J=7.0 Hz, 3H). ¹³C NMR(126 MHz, DMSO) δ 156.9, 154.3, 145.2, 140.1, 137.2, 135.0, 131.6,128.1, 127.5, 125.2, 124.8, 119.1, 112.5, 110.6, 94.2, 71.4, 63.0, 62.5,56.1, 36.2, 34.0, 15.0. HRMS (ESI⁺) Calcd for C₂₂H₂₂BrN₄O₃S⁺=501.0591,found 501.0590.

4-(3-Bromo-4-methoxyphenyl)-6-(methylamino)-3-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(13)

Compound 6a (105 mg, 0.244 mmol) was suspended in 1.60 ml of triethylorthoformate and 0.40 ml of glacial acetic acid were added dropwise. Thereaction vessel was immersed in in oil bath at 110° C., and the mixturewas stirred for 40 min. Conversion was judged complete by tlc(R_(f)=0.45 using hexanes, ethyl acetate 1:1). The mixture wasconcentrated to dryness, and the crude was suspended in 10 ml of EtOHand cooled down in ice/water bath. Sodium borohydrate (23.0 mg, 0.610mmol) was added in one portion and the reaction mixture was stirred for15 min, then the ice/water bath was removed and the mixture was stirredfor 1 h. Conversion was measured by TLC, using a mixture of hexanes,ethyl acetate 1:1, the product has R_(f)=0.30. Ethyl acetate (3 mL) wasadded and the mixture was concentrated under reduced pressure. The crudewas dissolved with ethyl acetate (20 mL) and 20 ml of 0.5 M aqueoussolution of acetic acid were added. The aqueous layer was extracted 3times with 40 ml of ethyl acetate. The combined organic layers weredried with brine and anhydrous sodium sulfate. The crude was purifiedwith automated flash chromatography, using a 16 g silica gel column anda mixture of hexane (A) and ethyl acetate (B) with the followinggradient 3 min 0% B, 10 min 50% B, 18 min 50% B, 33 min 100% B, 43 min100% B.

The title compound was isolated as colorless solid (62.0 mg), in 70%yield. Proton NMR (500 MHz, DMSO-d6) δ 13.07 (s, 1H), 7.55 (d, J=5.2 Hz,1H), 7.26 (d, J=3.2 Hz, 1H), 7.24 (d, J=2.2 Hz, 1H), 7.10 (dd, J=8.5,2.2 Hz, 1H), 7.07 (d, J=4.8 Hz, 1H), 7.03 (dd, J=5.0, 3.6 Hz, 1H), 6.99(d, J=8.5 Hz, 1H), 4.77 (s, 1H), 3.77 (s, 3H), 2.82 (d, J=4.7 Hz, 3H).¹³C NMR (126 MHz, DMSO) δ 159.5, 155.7, 154.1, 138.1, 133.0, 131.5,129.5, 128.0, 127.6, 127.4, 125.8, 120.7, 112.5, 110.3, 96.8, 57.6,56.1, 35.6, 28.1. HRMS (ESI⁺) Calcd for C₁₉H₁₆BrN₄O₂S⁺=443.0172, found443.0143.

2-Acetyl-6-amino-4-(3-bromo-4-methoxyphenyl)-3-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(14)

Compound 6a (50 mg, 0.11 mmol) was suspended in acetic anhydride (1 ml)and the mixture was stirred in a preheated oil bath at 110° C. for 30min. Upon complete conversion (as judged by TLC with hexanes, ethylacetate 1:1, R_(f)=0.48), the solvent was evaporated under reducedpressure, and the crude was purified by automated flash chromatographyusing a silica gel column (16 g) and a mixture of ethyl acetate (B) inhexanes (A), with the following gradient: 20 min 0 to 20% B, 70 min 24%B, 90 min 40% B. The title compound was isolated as a colorless solid in73% yield. Proton NMR (500 MHz, DMSO-d6) δ 7.54 (dd, J=5.1, 1.1 Hz, 1H),7.41 (d, J=2.2 Hz, 1H), 7.26 (d, J=2.2 Hz, 1H), 7.24 (d, J=2.2 Hz, 2H),7.16 (dd, J=3.7, 1.1 Hz, 1H), 7.02 (d, J=8.6 Hz, 1H), 6.99 (dd, J=5.1,3.7 Hz, 1H), 4.97 (s, 1H), 3.79 (s, 3H), 2.63 (s, 3H). ¹³C NMR (126 MHz,DMSO) δ 168.0, 158.3, 154.3, 147.5, 144.1, 137.0, 133.2, 131.7, 128.2,127.9, 127.8, 127.5, 119.5, 112.5, 110.7, 98.2, 58.8, 56.1, 35.0, 23.1.

4-(3-Bromo-4-methoxyphenyl)-3-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(15)

Compound 6a (50 mg, 0.11 mmol) was dissolved in anhydrous DMF (1 mL)under N2 atmosphere and the resulting pale yellow solution was cooleddown to −30° C. (internal temperature) in dry ice/acetone bath.Tert-butyl nitrite was added dropwise (30 μL, 0.25 mmol) (bright yellowsolution after the addition), and the mixture was stirred for 1 h, whilethe temperature reached 0° C. The cold bath was then removed and thereaction was stirred for an additional hour at room temperature. Uponcomplete conversion (monitored by tlc and indicated by the disappearanceof the spot corresponding to the starting material R_(f)=0.22 inhexanes, ethyl acetate 1:1). The reaction mixture was immersed in thecold bath and the internal temperature was reduced to −24° C. and thensodium borohydrate (13 mg, 0.35 mmol) was added in one portion, and thereaction mixture was stirred for one hour without controlling thetemperature, then the cold bath was removed and the reaction was stirredat room temperature for 1 hour. Ethyl acetate (3 mL) was added, and thesolvent was concentrated under reduced pressure. The crude was dissolvedin ethyl acetate (5 mL), water (5 mL) was added and the pH was adjustedto 7 with saturated aqueous solution of NH₄Cl. The aqueous layer wasextracted with ethyl acetate and the combined organic layers weretreated with brine and anhydrous sodium sulfate. The crude was purifiedwith automated flash chromatography using a 7 g silica gel column and amixture of hexanes (A) and ethyl acetate (B) with the followinggradient: 5 min 30% B, 10 min 55% B, 20 min 60% B. The title compoundwas isolated as a colorless solid in 31% yield. Proton NMR (500 MHz,DMSO-d6) δ 13.17 (s, 1H), 7.79 (d, J=1.1 Hz, 1H), 7.56 (d, J=5.1 Hz,1H), 7.37 (d, J=2.2 Hz, 1H), 7.24 (d, J=3.5 Hz, 1H), 7.20 (dd, J=8.5,2.2 Hz, 1H), 7.06-7.02 (m, 2H), 5.06 (d, J=1.1 Hz, 1H), 3.79 (s, 3H).¹³C NMR (126 MHz, DMSO) δ 155.1, 154.6, 151.6, 135.4, 133.6, 132.3,129.1, 128.8, 127.6, 127.6 (two peaks overlap, in fact the peak at 127.6is higher than the others and similar compounds have two peaks with veryclose chemical shifts) 126.1, 117.3, 112.7, 110.6, 95.9, 94.2, 56.2,35.7. HRMS (ESI⁺) Calcd for C₁₈H₁₃BrN₃O₂S⁺=413.9906 (100.0%), 415.9886(97.3%) found 413.9901 (100.0%), 415.9879 (97.3%).

Those skilled in the art will recognize that numerous modifications canbe made to the specific implementations described above. Theimplementations should not be limited to the particular limitationsdescribed. Other implementations may be possible.

It is intended that that the scope of the present methods andcompositions be defined by the following claims. However, it must beunderstood that this disclosure may be practiced otherwise than isspecifically explained and illustrated without departing from its spiritor scope. It should be understood by those skilled in the art thatvarious alternatives to the embodiments described herein may be employedin practicing the claims without departing from the spirit and scope asdefined in the following claims.

We claim:
 1. A method for treating a patient having a disease mediatedby a USP7 malfunction comprising the step of administering atherapeutically effective amount of one or more compounds having aformula

or a pharmaceutically acceptable salt thereof, wherein R¹ is hydrogen,an alkyl or an acyl; R² is a heterocyclyl, cycloalkyl, cycloalkenyl,cycloheteroalkyl, cycloheteroalkenyl, aryl, heteroaryl, arylalkyl,arylalkenyl, or arylalkynyl, each of which is optionally substituted;R³, R⁴, R⁶, and R⁷ represent five substituents each independentlyselected from the group consisting of hydrogen, halo, azido, cyano,nitro, hydroxy, amino, thio, and derivatives thereof, and an acyl,alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, oxyalkyl,heteroalkynyl, a heterocyclyl, cycloalkyl, cycloalkenyl,cycloheteroalkyl, cycloheteroalkenyl, aryl, arylalkyl, arylalkenyl, andarylalkynyl, each of which is optionally substituted; or any twoadjacent substituents that are taken together with the attached carbonsto form an optionally substituted heterocycle and each of othersubstituents is defined as above; one or more of R³, R⁴, R⁶, and R⁷ arenot hydrogen; R⁵ is —OR, wherein R is an alkyl; and R⁸ is cyano or acarboxy ester, and one or more carriers, diluents, or excipients, to apatient in need of relief from said disorder.
 2. The method according toclaim 1, wherein said disease mediated by a USP7 malfunction comprisesinflammations, cancers, and immunological disorders.
 3. (You may want tolist here some specific examples of the diseases mediated by a USP7malfunction, prostate cancer, lung cancer, multiple myeloma) . . . . 4.The method according to claim 1, wherein R¹ is hydrogen, an acyl or analkyl.
 5. The method according to claim 1, wherein R² is an optionallysubstituted 5- or 6-membered-ring heterocycle.
 6. The method accordingto claim 5, wherein R² is


7. The method according to claim 1, wherein R¹ is hydrogen, an acyl oran alkyl; and R² is


8. The method according to claim 1, wherein R⁴ or R⁶ is an alkyl, halo,or haloalkyl.
 9. The compound according to claim 1, wherein R⁵ isoxyalkyl.
 10. The method according to claim 1, wherein R⁵ is oxyalkyl;and R⁶ is halo, alkyl, cyano, or haloalky.
 11. The method according toclaim 1, wherein R⁴ is hydrogen; R⁵ is oxyalkyl; and R⁶ is halo, alkyl,cyano, or haloalky.
 12. The method according to claim 1, wherein R² isan optionally substituted heterocycle; R⁴ is hydrogen; R⁵ is oxyalkyl;and R⁶ is halo, alkyl, cyano, or haloalkyl.
 13. The method according toclaim 1, wherein R¹ and R⁴ are hydrogen; R² is an optionally substitutedheterocycle; R⁵ is oxyalkyl; and R⁶ is halo, alkyl, cyano, or haloalky.14. The method according to claim 1, wherein R⁴ or R⁶ is an alkyl, halo,or haloalkyl, and R⁵ is an oxyalkyl or aminoalkyl.
 15. The methodaccording to claim 1, wherein R⁸ is a carboxy ester.
 16. The methodaccording to claim 1, wherein said compound is


17. The method according to claim 1, wherein said compound is


18. The method according to claim 1, wherein said compound is


19. A method for treating diseases mediated by USP7 malfunction, such asinflammation, cancer and immunological disorders, comprising the step ofadministering a therapeutically effective amount of a compound offormula (I) according to claim 1 in combination with one or more othercompounds of the same or different mode of action, and one or morecarriers, diluents, or excipients, to a patient in need of relief fromsaid disorder. 20.